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.

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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.

Recessive dystrophic epidermolysis bullosa (RDEB), a rare skin blistering disorder, is caused by loss-of-function mutations in the gene encoding type VII collagen (C7), COL7A1. The main phenotype in RDEB is compromised skin architecture and fragility, resulting in constant wounding and excessive scarring. Nonfunctional C7 results in defective anchoring fibrils, which provide adhesion between the epidermis and dermis. In this study we are interested in premature termination codon (PTC) mutations in COL7A1. PTCs are associated with mRNA instability and nonsense mediated mRNA decay (NMD) as well as early termination of protein translation, thus leading to non-functional protein. We evaluate a read-through approach for overcoming PTC mutations in COL7A1 as potential therapy for RDEB. Reading-through PTCs allows for insertion of an amino acid at the mutation site and synthesis of full-length protein. Several drugs have been reported for their read-through ability but their efficacy is gene dependent. Here we evaluate an FDA approved drug-amlexanox-and demonstrate that it induces full-length C7 synthesis in patient derived RDEB keratinocytes and fibroblasts in a codon sequence specific manner. From six mutant alleles tested, four responded to amlexanox treatment. Treated RDEB cells showed 3-to 14-fold increase of C7 synthesis by western blot and 2-to 5-fold upregulation of COL7A1 mRNA levels, measured by qPCR. In our system read-through correlated with UPF1 phosphorylation state e the main factor in NMD. In conclusion, our study shows that amlexanox induces PTC read-through and synthesis of fulllength C7 in RDEB cells and upregulates COL7A1 transcript levels. Our results suggest amlexanox as potential therapy for RDEB patients harboring PTC mutations.