title: Ex vivo culture of primary human colonic tissue for studying transcriptional responses to 1α,25(OH)2 and 25(OH) vitamin D.
abstract: 1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] is a steroid hormone derived from circulating 25(OH) vitamin D [25(OH)D] with chemopreventive effects in colorectal cancer. 1α,25(OH)2D3 acts through transcriptional mechanisms; however, our understanding of vitamin D transcriptional responses in the colon is derived from studies in transformed cancer cell lines which may not represent responses in normal healthy tissue. Here, we describe the optimization of an ex vivo culture model using primary colonic biopsy samples for studying short-term transcriptional response induced by 1α,25(OH)2D3 and 25(OH)D treatment. Colon biopsy samples from healthy subjects were maintained in primary culture and treated in parallel with 100 nM 1α,25(OH)2D3 or 62.5 nM 25(OH)D and vehicle control (ethanol). Viability was assessed using histology and enzymatic assays. Genome-wide transcriptional responses to 1α,25(OH)2D3 were assessed and expression of 25(OH)D targets CYP27B1 and CYP24A1 were measured by real time PCR. We show that ex vivo culture of colonic tissue remains viable for up to 8 h. The largest number of differentially expressed genes in response to 1α,25(OH)2D3 was noted after 6 h (n = 120). As proof of concept, the top upregulated gene was CYP24A1, a well-established vitamin D-responsive gene. With 25(OH)D treatment, mRNA expression of CYP27B1 was significantly increased after 1 h, while expression of CYP24A1 was greatest at 8 h. Ex vivo culture can be used to assess short-term transcriptional responses to 1α,25(OH)2D3 and 25(OH)D in primary tissue from human colon. Future studies will address interindividual differences in transcriptional responses.
authors.affiliation: Section of Gastroenterology, Department of Medicine, University of Chicago Medical Center, Chicago, Illinois.
authors.forename: Brandon
authors.initials: B
authors.lastname: Mapes
authors.forename: Meredith
authors.initials: M
authors.lastname: Chase
authors.forename: Ellie
authors.initials: E
authors.lastname: Hong
authors.forename: Anton
authors.initials: A
authors.lastname: Ludvik
authors.forename: Katy
authors.initials: K
authors.lastname: Ceryes
authors.forename: Yong
authors.initials: Y
authors.lastname: Huang
authors.forename: Sonia S
authors.initials: SS
authors.lastname: Kupfer
mesh_terms: D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism; D003106:Colon / Q000378:metabolism*; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D046509:Tissue Culture Techniques / Q000379:methods; D014158:Transcription, Genetic / Q000235:genetics*; D015854:Up-Regulation / Q000235:genetics; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D065668:Vitamin D3 24-Hydroxylase / Q000235:genetics / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; C585549:CYP24A1 protein, human; D065668:Vitamin D3 24-Hydroxylase; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase
keywords: colon; primary tissue culture; transcriptional response; vitamin D