Xenobiotics such as medicines distinctly affect the expression of different genes as well as modulate signaling pathways in cells. One of the most important pathways, which activate processes such as proliferation, differentiation, inflammation and more, is the Wnt signaling pathway. Wnt factors can activate several signaling pathways such as the main strictly related to β-catenin -  the canonical and also two non-canonical, namely the Wnt/Ca²⁺ dependent and the planar cell polarity pathway. All of these start when a specific Wnt molecule binds to a frizzled receptor on the cell membrane and activates the dishivelled protein. Cyclosporine A is a immunosuppressant drug which acts as a calcineurine inhibitor, which regulates gene expression via NF-AT transcription factor which is also a part of  the non-canonical Wnt/Ca²⁺ dependent signaling pathway. Thus, the aim of our study was to estimate if cyclosporine can affect the Wnt signaling in healthy dermal fibroblasts.

Normal human dermal fibroblasts (NHDF, CC-2511, Lonza) were cultured in the liquid growth medium (Lonza, Basel, Switzerland) in standard conditions. After the confluence state there was added cyclosporine A (Sigma Aldrich) at a concentration  of 100 ng/ml. RNA had been extracted from cells after 8, 24, 48 hours by the use of TRIZOL® reagent accordingly to the producers protocol (Invitrogen Life Technologies, USA). The extracted RNA was purified and next used to synthesize cDNA and cRNA which was hybridized with the HG-U133A_ 2.0 Affymetrix microarray accordingly to the protocol (Affymetrix Inc. California, USA). Then the array was scanned with GeneArray Scanner 3000 7G (Affymetrix Inc.) obtaining fluorescence signal of 22277 mRNA. Statistical analysis were performed by the use of GeneSpring software (Agilent Technologies, USA), and only p < 0.05 were accepted.

We performed an ANOVA test for all culture times comparing them to the control without cyclosporine A and obtained 146 mRNA. The differences between all three culture times were checked with t test. In results there were only 9 mRNA common for all culture times. But also we noticed changes of Wnt molecules in different times.

After 24 h of human dermal fibroblasts exposition to cyclosporine A the Wnt5A expression had been over-expressed which suggest that via this pathway are trying to stimulate NF-AT signaling.

This work was supported by Medical University of Silesia grants KNW-1-060/K/3/0 and KNW-1-013/N/3/0.

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