Cdx-2 polymorphism in the vitamin D receptor gene (VDR) marks VDR expression in monocyte/macrophages through VDR promoter methylation
Caudal-type homeobox protein 2 (CDX-2) is an intestine-specific transcription factor (TF), with a polymorphic binding site (Cdx-2, rs11568820, A/G) in the vitamin D receptor gene (VDR). The molecular mechanism underlying Cdx-2 association with conditions like osteoporosis, which depends on intestinal VDR expression and calcium absorption, is believed to be due to higher affinity of CDX-2 for the ancestral A allele compared to the G allele. However, it is unclear why the polymorphism is associated with diseases like tuberculosis, which is dependent on VDR expression in immune cells that do not express CDX-2. This study aimed to explain Cdx-2 variant association with immune-related conditions. We hypothesised that the effect of Cdx-2 polymorphism on VDR expression in monocytes/macrophages, devoid of the CDX-2 TF, is indirect and dependent on circulating 25(OH)D3 and VDR methylation. Primary monocyte/macrophages from healthy donors (n = 100) were activated though TLR2/1 elicitation. VDR mRNA and 25(OH)D3 were quantified by RT-qPCR and LC-MS/MS, respectively. Genotyping and methylation analysis were done by pyrosequencing. AA vs. AG/GG showed reduced levels of 25(OH)D3 (P < 0.010), higher VDR promoter methylation (P < 0.050) and lower VDR mRNA induction (P < 0.050). Analysis of covariance confirmed that the effect of Cdx-2 variants depends primarily on VDR methylation. Thus, VDR methylation may confound association studies linking VDR polymorphisms to disease.
KeywordsVDR Cdx-2 Vitamin D 25-Hydroxyvitamin D3 DNA methylation
caudal-type homeobox protein 2
polymorphism in VDR
transcription factor binding to “GATA” motif
- Pam3CSK4 N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysyl x 3HCL
a synthetic triacylated lipopeptide that mimics the acylated amino terminus of bacterial lipopolysaccharide
Toll-like receptor 2/1 heterodimer
vitamin D receptor
We thank Miss TJ Jeffery, Dr. FF Asani and Mr. DS Saccone for their contribution to sample collection and data processing.
LB was the principal investigator, project leader and budget owner. VM acquired and analysed the data under the guidance of LB. Both LB and VM wrote the article. Both authors approved the final version of the manuscript.
This work was supported by grants to LB; the National Research foundation of South Africa (NRF, Grant No. 81774) and the Cancer Association of South Africa (CANSA). The funders played no role in the study design or in the collection and analysis of data.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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