ECE2017 Eposter Presentations: Interdisciplinary Endocrinology Nuclear receptors and Signal transduction (7 abstracts)
Vitamin D receptor (VDR) signalling can be affected with a variety of compounds, both endo- or exogenous. However, any disruption of VDR transcriptional activity can exert severe physiological or pathophysiological outcomes. It is therefore of a great importance to have a reliable in vitro screening technique that would provide a tool for efficient identification of agonists and activators of human VDR. In the current work, we described two novel human reporter cell lines IZ-CYP24 and IZ-VDRE, constructed for the purpose of VDR transcriptional activity detection. Parental human adenocarcinoma cell line LS 180 was stably transfected with a reporter construct CYP24_minP-pNL2.1[Nluc/Hygro] containing a sequence from the promoter region of human CYP24A1 gene (IZ-CYP24), or VDREI3_SV40-pNL2.1[Nluc/Hygro] containing three copies of VDREI from the promoter region of human CYP24A1 gene (IZ-VDRE), respectively. Both our cell lines remained fully functional in the cell culture for more than 2 months (corresponding to 30 passages) and even after cryopreservation. Luciferase inductions ranged from 10-fold to 25-fold (RLU 106107) and from 30-fold to 80-fold (RLU 103104) in IZ-VDRE and IZ-CYP24 cells, respectively. Time-course analyses revealed the possibility to detect VDR activators as soon as after 8 hours of incubation. Both our cell lines were highly selective towards VDR agonists, no cross-talk with retinoids, thyroids or steroid was observed. As a proof of concept, the effect of 13 human interleukins on VDR transcriptional activity was examined. Luciferase assays showed an inhibition of VDR transcriptional activity by interleukin-4 and interleukin-13, reaching approximately 60% of calcitriol-induced luciferase signal in concentrations about 1 ng/ml after 24 h of incubation in IZ-CYP24 cell line. Similar results were obtained from expression analyses of human CYP24A1 mRNA. Taken together, both of these cell lines provide a tool for reliable, high-throughput and selective identification of VDR ligands, with possible implications in toxicological and environmental studies.
20 May 2017 - 23 May 2017