Introduction. Localized scleroderma is a rare autoimmune disorder characterized by a thickening of the skin due to the overdeposition of collagens and other proteins in the dermal extracellular matrix. Matrix metalloproteinases (MMPs) is a family of proteolytic enzymes, playing a crucial role in the catabolism of collagens, maintaining the protein composition of the dermis. Alterations in MMPs activity and expression contribute to the development of the disease. Aim. To investigate changes in the expression of secreted matrix metalloproteinases (MMP-1, MMP-2, MMP-3, and MMP-9) in scleroderma lesional skin using quantitative polymerase chain reaction to identify potential therapeutic targets. Materials and methods. Biopsies of lesional and unaffected skin were obtained from patients with scleroderma. PCR was used to assess changes in the expression of MMPs. Results. The analysis of experimental data revealed significant changes in the expression of MMPs: the increased levels of MMP-2 (3.379 ± 1.177) and MMP-9 (4.471 ± 1.836), and the decreased levels of MMP-1 (0.169 ± 0.036) and MMP-3 (0.240 ± 0.086). The correlation analysis revealed a strong negative correlation ( r = –0.93) between the expression of MMP-1 and the area of scleroderma lesional skin. The computer analysis suggested that changes in the expression of MMPs may contribute to the development of skin lesions as a factor. Conclusions. The results of this study suggest that some MMPs may serve as biomarkers for assessing the severity of localized scleroderma and as potential molecular targets for therapeutic intervention.

Matrix metalloproteinases are a group of proteolytic enzymes that are important for the maintenance of skin homeostasis.The aim of this paper was to design and clone the sequence encoding scrambled small-hairpin RNA (shRNA) that can be used as a negative control in silencing the gene of human gelatinase B. Alternative sequences of shRNA were compared using the online tools "Blastn", "Oligo Calc" and "Palindromic sequences finder".Double stranded DNA encoding the selected shRNA was obtained by annealing of two complementary single stranded oligonucleotides.Commercial T4 DNA ligase and restriction endonucleases BamH1 and EcoRI were used to clone the annealed oligonucleotide into the expression vector pGPV-17019250.Thus, we designed Евразийский Союз Ученых (ЕСУ) #5( 62), 2019 scrambled shRNA that can be used as a counterpart of specific shRNA in silencing the gene of human gelatinase B and obtained the expression vector pGPV-17019250-KGB for its expression in cultured human cells.

Matrix metalloproteinases (MMPs) are important for the pathogenesis of psoriasis and other autoimmune disorders. In the extracellular matrix, accumulation of proinflammatory cytokines, such as interleukin 17A (IL-17A), leads to induction of several MMPs, including MMP1. MMPs change the composition and other properties of the extracellular matrix. These changes facilitate tissue remodeling and promote the development of psoriatic plaques. The aim of this study was to explore how MMP1 silencing might influence the biological effects of IL-17A on migration and proliferation of human epidermal keratinocytes and the expression of genes involved in their division and differentiation. The experiments were performed with MMP1-deficient and control epidermal keratinocytes, HaCaT-MMP1 and HaCaT-KTR, respectively. Cell proliferation and migration were assessed by comparative analysis of the growth curves and scratch assay, respectively. To quantify cell migration, representative areas of cell cultures were photographed at the indicated time points and compared to each other. Changes in gene expression were analyzed by real-time PCR. The obtained results demonstrated that MMP1 silencing in the cells treated with IL-17A resulted in downregulation of MMP9 and -12, FOSL1, CCNA2, IVL, KRT14 and -17 as well as upregulation of MMP2, CCND1 and LOR. Moreover, MMP1 silencing led to a decrease in cell proliferation and an impairment of cell migration. Thus, MMP1-deficiency in epidermal keratinocytes can be beneficial for psoriasis patients that experience an accumulation of IL-17 in lesional skin. Knocking MMP1 down could influence migration and proliferation of epidermal keratinocytes in vivo, as well as help to control the expression of MMP1, -2, -9 и -12, CCNA2, CCND1, KRT14 and -17 that are crucial for the pathogenesis of psoriasis.

The hyperproliferative human epidermal keratinocytes HaCaT are often used in experimental studies of psoriasis. Recently, we reported that Th1 cytokines TNF, IFN-γ and IL17 exhibited an antiproliferative effect on HaCaT cells. The aim of this paper was to explore how knocking matrix metalloproteinase 1 (MMP1) down in epidermal keratinocytes affected their proliferation, migration and differentiation and be beneficial for psoriasis. MMP1-deficient and control cells were generated by lentiviral transduction. The enzyme activity of MMP1 was assessed by zymography. Changes in gene expression were evaluated by real-time PCR. Scratch assay was used to analyze changes in the migration rate. Comparative analysis of MMP1-deficient and control cells revealed that knocking MMP1 down led to downregulation of MMP1 and decreased its catalytic activity by 80-85%. It also reduced the cell migration rate. However, it did not affect the cell proliferation. At the molecular level, MMP1-deficiency influenced the expression of genes involved in the pathogenesis of psoriasis (MMP9, -12, CCNA2, CCND1 and KRT17). Moreover, MMP1-deficient cells treated with IFN-γ demonstrated higher expression levels of the late differentiation markers LOR and FLG, cytokeratins (KRT1, -10 and -14) as well as the proapoptotic genes DAPK1 and IRF8. In turn, a prolonged culturing of these cells in the presence of IFN-γ impaired their proliferation and led to a cell death. The obtained data suggest that MMP1 silencing causes a significant decline in migration rate of HaCaT cells and makes them susceptible to IFN-γ by shifting the balance between proliferation and differentiation toward differentiation. In this respect, MMP1 silencing could be a perspective treatment option for hyperproliferative skin disorders, such as psoriasis.

Matrix metalloproteinases play an important role in the pathogenesis of psoriasis. The aim of this paper was to explore the influence of MMP1 silencing with a specific shRNA on migration and proliferation of epidermal keratinocytes exposed to tumor necrosis factor, as well as changes in the expression of genes involved in their terminal differentiation. Changes in gene expression were analyzed by real-time PCR. The cell proliferation was assessed by comparative analysis of the growth curves. The cell migration was explored by scratch assay. To quantify cell migration, the representative areas of cell cultures were photographed in the equal periods of time and compared to each other. The obtained results demonstrated that an exposure of control cell line to tumor necrosis factor caused changes in the expression of several genes similar to ones that were previously observed in lesional psoriatic skin. Particularly, the expression of MMP9, IVL and KRT16 increased whereas the expression of LOR, KRT1 and-10—decreased. In contrast, MMP1-deficient cells treated with tumor necrosis factor exhibited higher levels of LOR, KRT1 and -10, as well as lower levels KRT16 and -17 compared to control cells treated with the same cytokines. Moreover, MMP1-deficient cells exhibited a lower level of CCNА2 and higher level of CCND1. In this respect, knocking MMP1 down resulted in a lower cell proliferation and migration rates of TNF-treated epidermal keratinocytes. In conclusion, this study demonstrated that MMP1 silencing with specific shRNA can be beneficial for psoriasis. We found that knocking MMP1 down has an antiproliferative effect on epidermal keratinocytes and partially normalizes the expression of cyclins CCNA2, and -D1, as well as the genes involved in the terminal differentiation of this kind of cells (LOR, KRT1, -10, -16 and -17).

Matrix metalloproteinases are crucial for the maintenance of skin homeostasis, wound healing, and fueling the inflammatory process. In psoriasis, matrix metalloproteinases contribute to epidermal remodeling. They also influence the composition of extracellular matrix and intercellular interactions. Moreover, matrix metalloproteinases are important for vasodilation of dermal microcapillaries. The aim of this paper is to clarify whether knocking matrix metalloproteinase 1 down in epidermal keratinocytes can be beneficial for psoriasis. Lentiviral transduction was used to obtain the cell lines HaCaT-IK and HaCaT-KTR that expressed shRNA specific to matrix metalloproteinase 1 and scrambled shRNA, respectively. Changes in gene expression were analyzed by qPCR. The enzyme activity of matrix metalloproteinase 1 was assessed by zymography. Scratch assay was used to assess migration in transduced cells. Comparative analysis of HaCaT-IK and HaCaT-KTR revealed that MMP1 silencing downregulated matrix metalloproteinase 1 and decreased the enzyme activity of the named enzyme in 7.5 and 4 times, respectively. However, MMP1 silencing changed neither the expression of homologous genes (MMP2, -9 and -12) nor proliferation rate of the transduced cells. Surprisingly, MMP1-silencing changed the expression of several genes that are essential for pathogenesis of psoriasis. Particularly, MMP1-silencing caused downregulation of CCNA2 (0.54 ± 0.14) and KRT17 (0.58 ± 0.18). It also led to induction of LOR (2.08 ± 0.16), FLG (3.29 ± 0.35), KRT1 -5 and -10 (2.06 ± 0.20, 1.50 ± 0.22 and 2.09 ± 0.10, respectively). Finally, MMP1-silencing suppressed cell migration on collagen. In conclusion, MMP1 silencing in human epidermal keratinocytes causes changes in expression of LOR, FLG KRT1, -5, -10 and -17 that can be beneficial for psoriasis.

Matrix metalloproteinases play an important role in maintaining skin homeostasis, promote wound healing, and are involved in triggering inflammation. They are implicated in the structural changes occurring in the epidermis of psoriatic patients and also facilitate infiltration of the skin by immune cells by regulating permeability of dermal capillaries. In this light, control over the enzymatic activity of matrix metalloproteinases is crucial for a successful treatment outcome in patients with psoriasis. The aim of this work was to investigate the effect of RNA interference on the progression of psoriasis by targeting interstitial collagenase of epidermal keratinocytes. As part of the experiment, the latter were transduced with lentiviral particles that encode small hairpin RNA. Gene expression was measured by real time polymerase chain reaction. Enzymatic activity was measured by zymography. RNA interference was found to lead to a 20- and 4-fold decrease in the expression and enzymatic activity of interstitial collagenase, respectively. Expression of homologous genes (MMP2, -9 and -12) changed insignificantly. In contrast, there were marked changes in expression of cytokeratin (KRT1: 16.89 +/- 0.97; KRT14: 2.36 +/- 0.19; KRT17: 0.12 +/- 0.01; KRT18: 0.56 +/- 0.02), involucrin (0.79 +/- 0.11) and filaggrin (6.99 +/- 0.97). Besides, RNA interference caused a significant decline in cell migration rates, although it did not affect cell proliferation. Thus, small hairpin RNAs targeting interstitial collagenase are potentially therapeutic for psoriatic patients due to their ability to regulate expression of genes implicated in psoriasis (IVL, FLG, KRT1, -14 -17, and -18).

Psoriasis is an immune-mediated skin condition with genetic predisposition. The gene that encodes interstitial collagenase MMP1 is upregulated in lesional psoriatic skin and downregulated in the skin in the period of disease remission. Our aim was to analyze the data on MMP1 expression, secretion and activation to characterize MMP1 as a possible candidate gene for gene therapy of psoriasis. Immunohistochemistry was used to detect MMP1 in psoriatic skin lesions. Changes in gene expression were detected by qPCR. Cell migration was assessed by scratch assay. Cell proliferation was estimated by comparing the cell growth in control cells and cells treated with proinflammatory factors (IL17, TNF, IFNG and S100A7). Our results suggest that MMP1 is among three other matrix metalloproteinases differentially expressed in psoriatic skin. We also found that MMP1 levels decline after a course of PUVA therapy. Moreover, higher MMP1 levels have been observed in psoriatic plaques and the blood serum of psoriatic patients. In addition, we were able to induce MMP1 by treatment of HaCaT with the proinflammatory factors IL17, TNF, IFNG and S100A7 and demonstrate that higher MMP1 levels in cells are associated with faster migration and higher proliferation rates. We conclude that MMP1 could be reconsidered as a treatment option for psoriasis if negative consequences were taken in account and necessary precautions were applied.

Proinflammatory cytokines TNF, IFNG, and IL17 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 IFNG. 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.

Провоспалительные цитокины: фактор некроза опухолей (ФНО), интерферон (ИФНГ) и интерлейкин 17 (ИЛ-17) – играют важную роль в патогенезе псориаза. Активация эпидермальных кератиноцитов этими цитокинами изменяет процесс дифференцировки клеток, что является основной причиной их гиперпролиферации в коже больных. Иммортализованную линию кератиноцитов человека HaCaT часто используют в качестве модельной системы для изучения молекулярных механизмов патогенеза псориаза. Установлено, что обработка клеток HaCaT провоспалительными цитокинами приводит к снижению пролиферации этих клеток. Анализ генной экспрессии выявил группу из 12 генов, экспрессия которых повышена при псориазе, но снижена в клетках HaCaT после их обработки цитокинами. Выявленные гены играют важную роль в процессе репликации ДНК и входят в состав двух других, более обширных групп генов с измененной экспрессией, участвующих в регуляции клеточного цикла. Полученные в ходе данной работы результаты свидетельствуют о том, что культура клеток HaCaT имеет существенное ограничение в качестве модельной системы псориаза: обработка HaCaT провоспалительными цитокинами не приводит к увеличению пролиферации клеток. Эту особенность необходимо принимать во внимание при изучении влияния новых лекарственных препаратов на пролиферацию клеток.

This review summarizes the existing knowledge regarding the role of receptor for advanced glycation end products in pathogenesis of psoriasis. This receptor plays a crucial role in the inflammatory response. By interacting with multiple ligands and activating several signaling mechanisms, receptor for advanced glycation end products regulates gene expression via a group of well-characterized transcription factors, such as NFkB and AP1. 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 promotes the development of psoriasis. 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 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 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 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.