Vitamin D Receptor Gene Expression and Function in a South African Population: Ethnicity, Vitamin D and FokI

Abstract

Polymorphisms of the vitamin D receptor gene (VDR) have been associated inconsistently with various diseases, across populations of diverse origin. The T(f) allele of the functional SNP FokI, in exon 2 of VDR, results in a longer vitamin D receptor protein (VDR) isoform, proposed to be less active. Genetic association of VDR with disease is likely confounded by ethnicity and environmental factors such as plasma 25(OH)D3 status. We hypothesized that VDR expression, VDR level and transactivation of target genes, CAMP and CYP24A1, depend on vitamin D, ethnicity and FokI genotype. Healthy volunteers participated in the study (African, n = 40 and White, n = 20). Plasma 25(OH)D3 levels were quantified by LC-MS and monocytes cultured, with or without 1,25(OH)2D3. Gene expression and protein level was quantified using qRT-PCR and flow cytometry, respectively. Mean plasma 25(OH)D3 status was normal and not significantly different between ethnicities. Neither 25(OH)D3 status nor 1,25(OH)2D3 supplementation significantly influenced expression or level of VDR. Africans had significantly higher mean VDR protein levels (P<0.050), nonetheless transactivated less CAMP expression than Whites. Genotyping the FokI polymorphism by pyrosequencing together with HapMap data, showed a significantly higher (P<0.050) frequency of the CC genotype in Africans than in Whites. FokI genotype, however, did not influence VDR expression or VDR level, but influenced overall transactivation of CAMP and 1,25(OH)2D3-elicited CYP24A1 induction; the latter, interacting with ethnicity. In conclusion, differential VDR expression relates to ethnicity, rather than 25(OH)D3 status and FokI genotype. Instead, VDR transactivation of CAMP is influenced by FokI genotype and, together with ethnicity, influence 1,25(OH)2D3-elicited CYP24A1 expression. Thus, the expression and role of VDR to transactivate target genes is determined not only by genetics, but also by ethnicity and environment involving complex interactions which may confound disease association.

Figure 1. Ethnicity influenced VDR mRNA and protein level.

In vitro VDR expression (A) and protein level (B) were quantified in monocyte-macrophages from healthy Africans (grey, n = 40) and Whites (white, n = 20), using RT-qPCR and flow cytometry respectively. Monocytes were analysed directly after isolation (Basal), or cultured for 24 h with 10 nM 1,25(OH)₂D₃ or vehicle control. Box plots show data distribution: boxes illustrate 50% of cases or interquartile range (IQR, 25th to 75th percentile); horizontal lines, median; whiskers, 1.5 IQR from box, or minimum or maximum values if no case has a value in that range. Africans displayed more variance than Whites as illustrated by the distribution IQRs and outliers (Δ, 1.5 to 3 IQR from box) and extreme outlier (♦, >3 IQR from box). Approximately 95% of the data lie between whiskers. Two-way ANOVA showed an overall, significant main effect for ethnicity; Africans having lower VDR mRNA (P<0.050) but higher protein level (P<0.001) compared to Whites. Fisher's LSD test showed a significantly higher mean VDR protein level in Africans compared to Whites under all conditions (* P<0.050). VDR mRNA data was ln-transformed to meet the assumptions of parametric statistical tests.

Figure 2. 1,25(OH)₂D₃-elicited transactivation of target gene CAMP by VDR is influenced by ethnicity.

Box plots illustrate expression of VDR target genes, CAMP (A) and CYP24A1 (B). Data is differentiated by ethnicity: Africans (grey, n = 40) and Whites (white, n = 20). Gene expression was quantified in monocyte-macrophages from healthy individuals using RT-qPCR. In vitro 1,25(OH)₂D₃ supplementation significantly induced CAMP and CYP24A1 expression in both Africans and Whites relative to the vehicle control level (P<.050, P<0.010, P<0.001). Ethnicity had a significant main effect on CAMP (P<0.010), but not CYP24A1 mRNA level. Significantly higher mean CAMP mRNA level was observed in Whites compared to Africans after 1,25(OH)₂D₃ supplementation (* P<0.050). CAMP and CYP24A1 mRNA data was ln-transformed to meet the assumptions of parametric statistical analysis. Outliers are defined in legend for Fig. 1.

Figure 3. 25(OH)D₃ status of Africans and Whites of the Gauteng Province of South Africa is normal and not significantly different.

The mean plasma 25(OH)D₃ status of Africans (84.4 nmol/L, n = 30) and Whites (92.4 nmol/L, n = 14), quantified using LC-MS, was normal according to the IOM recommendations and not significantly different between the two ethnic groups.

Figure 4. The impact of increasing in vitro 1,25(OH)₂D₃ supplementation on CAMP expression.

Error-bar plots illustrate the mean level of CAMP expression. Data is differentiated by ethnicity: Africans (black, n = 4) and Whites (white, n = 4). Gene expression was quantified in monocyte-macrophages from healthy individuals using RT-qPCR before (basal) and after 24 h of in vitro 1,25(OH)₂D₃ supplementation at increasing concentrations (10 nM, 50 nM, and 100 nM). The trend for higher CAMP in Whites and lower CAMP in Africans at 10 nM, appears to be reversed at higher levels of 1,25(OH)₂D₃ supplementation (50 nM and 100 nM). Error bars display the LSD for each data set.

Figure 5. Individual-specific response to increasing in vitro 1,25(OH)₂D₃ supplementation is 25(OH)D₃ status-independent.

Dots illustrate the mean level of CAMP expression per individual for Whites (A, 1–4) and Africans (B, 5–8). The 25(OH)D₃ status, as well as the 1,25(OH)₂D₃-mediated fold change in expression level relative to the control is shown for each individual (untransformed data). Gene expression was quantified in monocyte-macrophages from healthy individuals using RT-qPCR before (basal) and after 24 h of in vitro 1,25(OH)₂D₃ supplementation at increasing concentrations (10 nM, 50 nM, and 100 nM). Plasma 25(OH)D₃ status was quantified using LC-MS. A trend towards higher CAMP level is present at 10 nM 1,25(OH)₂D₃ for Whites, while Africans showed marginally increased CAMP expression at 50 nM 1,25(OH)₂D₃ concentrations. No trend between plasma 25(OH)D₃ status and in vitro response to increasing concentrations of 1,25(OH)₂D₃ is present.

Figure 6. FokI influence VDR function, but not expression.

Error-bar plots illustrate the mean level of VDR expression (A), VDR protein (B), CAMP expression (C) and CYP24A1 expression (D) differentiated by FokI genotype (CC and CT/TT). Data was analysed combining ethnicity (i and ii, grey dots, n = 57) or differentiating ethnicity (iii and iv, Africans [n = 38], black dots and Whites [n = 19], white dots). Data was further analysed combining (overall, i and iii) or separating treatments (ii and iv). VDR expression and protein level was not significantly influenced by FokI genotype. Combining ethnicity and treatment (overall), CAMP mRNA level was significantly higher in the CT/TT genotypes compared to the CC genotype (* P<0.050). 1,25(OH)₂D₃-elicited induction of CYP24A1 mRNA was significant in Africans with the CC genotype (P<0.010, n = 26) and in Whites with the CT/TT genotypes (P<0.050, n = 12). Error bars display the LSD for each data set with Bonferroni correction. All significances indicated withstood Bonferroni correction.