Calcium and 1,25-dihydroxyvitamin D3 modulate genes of immune and inflammatory pathways in the human colon: a human crossover trial

Abstract

Background: A high dietary calcium intake with adequate vitamin D status has been linked to lower colorectal cancer risk, but the mechanisms of these effects are poorly understood.

Objective: The objective of this study was to elucidate the effects of a Western-style diet (WD) and supplemental calcium and/or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the colorectal mucosa.

Design: We conducted 2 crossover trials to define molecular pathways in the human colorectum altered by 1) a 4-wk WD supplemented with and without 2 g calcium carbonate/d and 2) a 4-wk WD supplemented with 1,25(OH)2D3 (0.5 μg/d) with or without 2 g calcium carbonate/d. The primary study endpoint was genome-wide gene expression in biopsy specimens of the rectosigmoid colonic mucosa. Serum and urinary calcium concentrations were also measured.

Results: Changes in urinary calcium accurately reflected calcium consumption. The WD induced modest upregulation of genes involved in inflammatory pathways, including interferon signaling, and calcium supplementation reversed these toward baseline. In contrast, supplementation of the WD with 1,25(OH)2D3 induced striking upregulation of genes involved in inflammation, immune response, extracellular matrix, and cell adhesion. Calcium supplementation largely abrogated these changes.

Conclusions: Supplementing 1,25(OH)2D3 to a WD markedly upregulated genes in immune response and inflammation pathways, which were largely reversed by calcium supplementation. This study provides clinical trial evidence of global gene expression changes occurring in the human colorectum in response to calcium and 1,25(OH)2D3 intervention. One action of 1,25(OH)2D3 is to upregulate adaptive immunity. Calcium appears to modulate this effect, pointing to its biological interaction in the mucosa. This trial was registered at clinicaltrials.gov as NCT00298545 Trial protocol is available at http://clinicalstudies.rucares.org (protocol numbers PHO475 and PHO554).

Keywords: 1,25-dihydroxyvitamin D3; Western diet; calcium; colon; gene expression.

© 2016 American Society for Nutrition.

Figures

FIGURE 1

Diagram of study organization. The figure represents an overview of the study design. All interventions were performed for 4 wk while subjects consumed a WD with or without supplemental calcium (as 2 g calcium carbonate/d) (study A) or a WD supplemented with vitamin D (as 0.5 μg calcitriol/d) with or without supplemental calcium (study B). There was a 4-wk “washout” period during which the subjects were advised by our nutritionists to return strictly to their prestudy diet. There was no run-in period for study B because the WD was found to be so similar to the subjects’ prestudy diets. WD, Western-style diet.

FIGURE 2

Heatmap of Gene Set Enrichment Analysis of canonical pathways. The WD induced small but significant upregulation of genes involved in immunity-modulated pathways such as interferon signaling or antigen processing and presentation. Supplementation with calcium partially reversed these changes toward baseline. The WD supplemented with vitamin D induced striking upregulation of genes involved in immune response and inflammation, extracellular matrix, and cell adhesion and cell cycle. Addition of calcium largely reversed these changes. Only the top significant pathways and categories are presented in the figure (FDR-q val < 0.005). For a full list of significantly enriched pathways and categories, please refer to Table 2 and Supplemental Table 4. FDR-q val, false discovery rate adjusted significance (q value); WD, Western-style diet.

FIGURE 3

Heatmap of Gene Set Enrichment Analysis of Transcriptional Factor targets. The Western-style diet supplemented with vitamin D induced significant upregulation of VDR target genes (VDR, FDR-q val = 0.09) and target genes of other transcription factors that have key roles in immune response, proliferation, and extracellular matrix function. Addition of calcium reversed these changes toward baseline. Note that only the top significant pathways and categories are presented in the figure (FDR-q val < 0.01). For a full list of significantly enriched pathways, please refer to Supplemental Table 5. FDR-q val, false discovery rate adjusted significance (q value); VDR, vitamin D receptor.