Profiling of Vitamin D Metabolic Intermediates toward VDR Using Novel Stable Gene Reporter Cell Lines IZ-VDRE and IZ-CYP24.
Variety of xenobiotics, including therapeutically used vitamin D analogues or environmental and alimentary endocrine disruptors, may interfere with vitamin D receptor (VDR) signaling, with serious physiological or pathophysiological consequences. Therefore, it is of topical interest to have reliable and efficient in vitro screening tools for the identification of agonists and activators of human VDR. We present here two novel stably transfected human reporter cell lines allowing rapid, high-throughput, and selective identification of VDR agonists and activators. Human colon adenocarcinoma cells LS180 were stably transfected with reporter plasmids CYP24_minP-pNL2.1[Nluc/Hygro] (IZ-CYP24 cells contain the -326/-46 sequence from the human CYP24A1 promoter) or VDREI3_SV40-pNL2.1[Nluc/Hygro] (IZ-VDRE cells contain three copies of vitamin D response elements VDRE-I from the human CYP24A1 promoter). Both cell lines remained fully functional for over two months in the culture and also after cryopreservation. Luciferase inductions ranged from 10-fold to 25-fold (RLU 10(6)-10(7)) and from 30-fold to 80-fold (RLU 10(3)-10(4)) in IZ-VDRE and IZ-CYP24 cells, respectively. Time-course analyses revealed that detection of VDR activators is possible as soon as after 8 h of incubation. Cell lines were highly selective toward VDR agonists, displaying no cross-activation by retinoids, thyroids, and steroids. As a proof of concept, we used IZ-VDRE and IZ-CYP24 cells for profiling analogues of vitamin D, and intermediates in vitamin D2 and vitamin D3 metabolic pathways against VDR transcriptional activity. The data obtained revealed significant activation of VDR not only by obligatory ligands calcitriol and ergocalcitriol but also by their precursors and degradation products.
Department of Cell Biology and Genetics, Faculty of Science, Palacky University , Slechtitelu 27, 783 71 Olomouc, Czech Republic.
Iveta
I
Bartonkova
Department of Cell Biology and Genetics, Faculty of Science, Palacky University , Slechtitelu 27, 783 71 Olomouc, Czech Republic.
Aneta
A
Grycova
Department of Cell Biology and Genetics, Faculty of Science, Palacky University , Slechtitelu 27, 783 71 Olomouc, Czech Republic.
Zdenek
Z
Dvorak
D045744:Cell Line, Tumor; D000072498:Cytochrome P450 Family 24 / Q000235:genetics*; D017930:Genes, Reporter; D006801:Humans; D018167:Receptors, Calcitriol / Q000378:metabolism*; D014807:Vitamin D / Q000378:metabolism*
D018167:Receptors, Calcitriol; D014807:Vitamin D; D000072498:Cytochrome P450 Family 24