Analysis of 1,25-dihydroxyvitamin D genomic action in human enteroids and colonoids reveals multiple regulatory effects of vitamin D in human intestinal physiology.

Abstract

Introduction: The intestine has molecular and functional diversity across the proximal-distal and the crypt-villus axes, so it is imperative to determine the common and compartment-specific molecular actions of vitamin D. However, very little work on vitamin D mediated gene regulation has been done in normal human intestine. Here, we examined the impact of 1,25-dihydroxyvitamin D (1,25(OH)₂D₃) on cultures of human intestinal epithelium derived from duodenum (Dd) and distal colon (Co) biopsies of 6 subjects per tissue.

Methods: Human enteroids and colonoids were cultured for 3 days to promote a stem cell phenotype (undifferentiated, Un) or to induce differentiation (Diff) and then treated with vehicle control or 1,25(OH)₂D₃ (100 nM). 24h following treatment enteroids/colonoids were collected, RNA was isolated and RNA-seq was performed using paired-end Illumina sequencing (analysis in R using DESeq2).

Results and discussion: RNA-seq analysis showed that VDR mRNA is present in all four cultures tested (DdUn, DdDiff, CoUn, CoDiff) and it is not altered by 1,25(OH)₂D₃ treatment, intestinal segment, or differentiation status. 1,25(OH)₂D₃ induced the classic intestinal target genes TRPV6, ATP2B1 and CYP24A1 in all four culture groups while S100G was induced only in DdDiff. While 63 genes were vitamin D regulated across all four cultures (55 up, 8 down), we found that vitamin D regulated subgroups of genes within Dd, Co, Un, or Diff groups as well as set of genes that were unique to each culture. Functional analysis revealed several vitamin D-enriched gene ontologies or pathways including those for xenobiotic/drug metabolism in all four cultures. In differentiated cultures vitamin D induced genes were enriched for functions like regulation of barrier function through regulation of Rho GTPases and metabolism of lipids while vitamin D downregulated genes in Un groups were enriched for activities like water transport. These results provide new insight into 1,25(OH)₂D₃ genomic action in the functionally distinct compartments and segments of human intestine and suggest multiple regulatory effects of vitamin D in human intestinal physiology.