Profiling of Vitamin D Metabolic Intermediates toward VDR Using Novel Stable Gene Reporter Cell Lines IZ-VDRE and IZ-CYP24

Abstract

The role of vitamin D receptor (VDR) in immune responses has been broadly studied and it has been shown that activated VDR alters the levels of some interleukins (ILs). In this study, we studied the opposite, i.e. whether 13 selected pro-inflammatory and anti-inflammatory ILs influence the transcriptional activity of human VDR. The experimental models of choice were two human stably transfected gene reporter cell lines IZ-VDRE and IZ-CYP24, which were designed to evaluate the transcriptional activity of VDR. The gene reporter assays revealed inhibition of calcitriol-induced luciferase activity by IL-4 and IL-13, when 1 ng/mL of these two compounds decreased the effect of calcitriol down to 60% of the control value. Consistently, calcitriol-induced expression of CYP24A1 mRNA was also significantly decreased by IL-4 and IL-13. The expression of VDR and CYP27B1 mRNAs was not influenced by any of the 13 tested ILs. These data suggest possible cross-talk between the VDR signalling pathway and IL-4-and IL-13-mediated cell signalling.

Targeting the Pregnane X Receptor Using Microbial Metabolite Mimicry

“…87021202) were purchased from European Collection of Cell Cultures (ECACC). Stably transfected gene reporter cell lines AZ-AHR, AZ-GR, IZ-VDRE, PZ-TR and AIZ-AR were as described elsewhere [12][13][14]47,48 . Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) or RPMI-1640 medium (cultivation of AIZ-AR cells) supplemented with 10% of fetal bovine serum, 100 U/mL streptomycin, 100 mg/mL penicillin, 4 mM L-glutamine, 1% non-essential amino acids, and 1 mM sodium pyruvate.…”

“…2c) and androgen receptor (AR) ( Supplementary Fig. 2d) using cell-based luciferase assays previously described 13,14 . Similarly, FKK5 or 6 did not induce constitutive androstane receptor (CAR) activity ( Supplementary Fig.…”

“…to assess cell cytotoxicity using the MTT assay as previously described 13,14 . The results show that the entire series of FKK compounds (up to 10 μM) do not significantly alter LS180 cell survival ( Supplementary Fig.…”

“…LS180 cells transiently transfected by lipofection (FuGENE HD Transfection Reagent) with pSG5-PXR plasmid along with a luciferase reporter plasmid p3A4-luc 10,11 , were used for assessment of PXR transcriptional activity. Transcriptional activity of AHR, GR, VDR, TR and AR were studied in stably transfected gene reporter cell lines AZ-AHR, AZ-GR, IZ-VDRE, PZ-TR and AIZ-AR, respectively [12][13][14]47,48 . Cells were seeded in 96-well plates, stabilized for 24 h, and Differences were tested using one-way ANOVA with Dunnett's post hoc test, p < 0.05, was considered significant (*).…”

Fine tuning of vitamin D receptor (VDR) activity by post-transcriptional and post-translational modifications

“…However, recent research brought the proof that vitamin D 2 is equally effective [16]. Moreover, binding affinities of 1,25(OH) 2 D 2 (ergocalcitriol) or 1,25(OH) 2 D 3 (calcitriol) to VDR or transactivation potencies on target genes are almost comparable [17, 18]. …”