25‐Hydroxyvitamin D Elicits Hormone‐Like Response in Intestinal Epithelium
Authors: Reynolds Carmen , Koszewski Nicholas , Horst Ronald , Beitz Donald , Goff Jesse 
Published: November 2023

Animal‐based foods such as eggs, chicken, and fish are known to contain 25‐hydroxyvitamin D (25D), the precursor for hormonal 1,25‐dihydroxyvitamin D (1,25D). Because digestion breaks down proteins that may sequester 25D in these foods, the molecule is presumed to be freely accessible by the intestinal epithelium. In this study, we hypothesized that orally consumed 25D can function bioactively in the intestines and elicit a hormonal response comparable to 1,25D. Our objective was to assess expression of cytochrome P450 24 A1 (CYP24) mRNA, an indicator of 1,25D action, after treatment of 1,25D or 25D both in vitro and in vivo.MethodsIn vitro assays were conducted with Caco2 and HT29 cells. Cells were incubated with media containing 10%, 1%, or 0.1% fetal bovine serum (FBS) and treated with ethanol, 25D or 1,25D for 18 hours before lysing. Expressed mRNA was analyzed for glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and CYP24 by QPCR. In vivo trials were conducted in mice that were orally gavaged with ethanol, 1,25D, or 25D. In preliminary trials, intestinal segments from 1,25D‐treated mice (6 hours) were analyzed for RNA expression of GAPDH and CYP24 by QPCR and CYP24 by RNA Scope in situ hybridization (RISH). Intestinal segments of 25D‐treated mice were qualitatively assessed for GAPDH and CYP24 by conventional PCR.ResultsCells incubated in media with 10% FBS responded to 1,25D with >2000‐fold greater response for CYP24 expression compared to controls with both Caco2 (P<0.001) and HT29 (P<0.001). No difference was observed with CYP24 expression in response to 25D compared to controls (P>0.3). Caco2 and HT29 cells were not affected by low serum media in control and 1,25D treatments. In Caco2 cells, 25D treatment in 1% and 0.1% FBS‐media induced a 1000‐fold (P<0.01) and 2000‐fold (P<0.01) greater expression than 25D treatment in 10% FBS‐media, respectively. Likewise, HT29 cells responded to low serum media with >100‐fold increase in CYP24 in both 1% (P<0.01) and 0.1% (P<0.01) FBS enriched media. It was apparent that serum in media sequesters 25D from the cells and that removing the serum allowed for 25D to elicit an upregulation of CYP24 similar to that of 1,25D treatments. In preliminary trials with mice, an oral gavage of 24 pmol 1,25D induced CYP24 expression in the duodenum 250‐fold greater compared to vehicle‐treated mice (n=3, P<0.001). RNA in situ hybridization identified localization of 1,25D response to be confined to intestinal epithelial cells. Though preliminary analysis of 25D‐treated mice is qualitative, it is apparent that 25D also induces expression of CYP24 in duodenums comparable with 1,25D treatments.ConclusionWe have demonstrated that 25D can elicit a response in CYP24 mRNA expression comparable to that of 1,25D treatment both in vitro and in vivo. Furthermore, we have shown that the response is confined to intestinal epithelium where cells are likely exposed to free 25D in digesta. Thus, 25D consumed in foods may hold bioactive functions that target the intestinal epithelium.Support or Funding InformationThis research was funding by NIH Grant CA 173628.