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American Journal of Physiology - Gastrointestinal and Liver Physiology logoLink to American Journal of Physiology - Gastrointestinal and Liver Physiology
. 2020 Jul 6;319(2):G253–G260. doi: 10.1152/ajpgi.00355.2019

Role of glucuronidated 25-hydroxyvitamin D on colon gene expression in mice

Carmen J Reynolds 1,✉, Nicholas J Koszewski 2, Ronald L Horst 3, Donald C Beitz 1, Jesse P Goff 2
PMCID: PMC7500262  PMID: 32628073

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Keywords: calcifediol, colon calcium absorption, glucuronic acid, 25-hydroxyvitamin D, vitamin D

Abstract

25-Hydroxyvitamin D3-3β-glucuronic acid (25OHD-Gluc) is produced in the liver and is a constituent of human blood and bile. Bacterial glucuronidases (GUS) in mammalian digestive microbiota cleave glucuronide conjugates, such as 25OHD-Gluc, and release the free aglycone (i.e., 25OHD) inside the intestinal lumen. We hypothesized that 25OHD-Gluc would elicit a VDR-dependent mRNA response in the colon after cleavage by gut microbiota. The activity of 25OHD-Gluc was investigated by measuring expression of cytochrome P450 24A1 (Cyp24) mRNA both in vitro and in vivo. In cell culture, Caco2 cells responded to 25OHD-Gluc, whereas HT29 cells did not. When coincubated with GUS, both cell lines elicited a robust response as indicated by a 5 Ct (32-fold) increase in Cyp24 mRNA. In vitamin D-sufficient mice, we found that both oral and subcutaneous administration of 1 nmol 25OHD-Gluc induced expression of Cyp24 mRNA in the colon whereas 25OHD did not. In contrast, 25OHD, but not 25OHD-Gluc, was active in the duodenum. When the jejunum was surgically ligated to block flow of digesta to the colon, neither oral nor subcutaneous administration of 2 nmol 25OHD-Gluc was able to induce expression of Cyp24 in the colon. Our findings suggest that 25OHD-Gluc, a vitamin D metabolite found in bile, induces VDR-mediated responses in the colon by crossing the apical membrane of the colon epithelium.

NEW & NOTEWORTHY We found that 25OHD-Gluc, an endogenously produced metabolite, is delivered to the colon via bile to induce vitamin D-mediated responses in the colon.

INTRODUCTION

Activation of the intestinal vitamin D receptor (VDR) mediates transcellular calcium absorption by promoting transcription of calcium channel, transport, and trafficking proteins. VDR is expressed in the colon (4, 5, 28, 39), but the mechanism by which its preferred substrate, 1,25-dihydroxyvitamin D3 [1,25(OH)2D], activates VDR in the colon is unclear. Previous studies by our laboratory have shown that oral or subcutaneous administration of exogenous 1,25(OH)2D at doses that greatly stimulate duodenal gene expression have little effect on colon gene expression. Instead, administration of the glucuronide conjugate 1,25-dihydroxyvitamin D3-25β-glucuronic acid (1,25(OH)2D-Gluc) successfully activates the VDR response in the colon. This targeted response occurs because 1,25(OH)2D-Gluc is inactive until it reaches the colon where bacterial β-glucuronidases (GUS) cleave the glucuronide moiety and liberate functional 1,25(OH)2D. The result is upregulation of VDR-mediated genes in the colon rather than the duodenum and with only minor increases in plasma 1,25(OH)2D concentrations (17, 22, 32). The same delivery mechanism occurs for plant-derived glycosides and a diglucuronide of 1,25(OH)2D that also induce the vitamin D response in the colon, thus further implicating the coordination between these conjugated 1,25(OH)2D molecules and the gut bacteria (20). Interestingly, endogenous production of 1,25(OH)2D-Gluc has been observed following intravenous administration of 1,25(OH)2D, but the low concentrations in blood and bile raise concern about the physiological relevance of endogenous 1,25(OH)2D-Gluc (2, 24, 25).

The major circulating metabolite of vitamin D is 25-hydroxyvitamin D (25OHD). The glucuronide of 25OHD, 25-hydroxyvitamin D3-3β-glucuronic acid (25OHD-Gluc), has recently been shown to be a product of the liver enzyme, UDP-glucuronosyltransferase 1A4 (40). Furthermore, 25OHD-Gluc has been observed in blood plasma (1–5 nM) and bile (detectable by mass spectrometry) without prior administration of the precursor, 25OHD (13, 40). We hypothesized that 25OHD-Gluc would be subject to cleavage by bacterial GUS to liberate 25OHD in the colon. The aglycone 25OHD is not only a precursor to 1,25(OH)2D, but also has agonistic actions on VDR when not bound to the vitamin D binding protein (DBP) (3, 27, 28, 33, 36, 38). The liberated 25OHD could then act as a ligand to stimulate the VDR-mediated response in the colon. To test our hypothesis, we first verified the activity of 25OHD-Gluc on vitamin D-dependent gene expression with two human colon adenocarcinoma cell lines, HT-29 and Caco2. Next, we evaluated VDR-mediated intestinal responses in mice by administering 25OHD-Gluc orally and subcutaneously and then measuring changes in mRNA expression of the vitamin D-dependent gene, cytochrome P450 24A1 (Cyp24), in both the duodenum and colon. Lastly, we blocked passage of digesta and bile beyond the small intestine by surgical ligation to discern if the mechanism by which 25OHD-Gluc upregulates colon gene expression requires passage down the digestive tract and entry across the apical membrane of colon epithelial cells versus raising blood concentrations of 25OHD to allow entry into colon cells across the basolateral cell membrane.

METHODS

Experimental Reagents

Vitamin D metabolites, including 25OHD, 25OHD-Gluc, 1,25(OH)2D, and 1,25(OH)2D-Gluc, were used in cell culture and mouse studies. Both 25OHD and 1,25(OH)2D were purchased from Sigma Aldrich (St. Louis, MO). The glucuronide conjugates 25OHD-Gluc (mol wt. 576.76) and 1,25(OH)2D-Gluc (mol wt. 592.76) were synthesized from their respective metabolites in our laboratory and diluted in 100% EtOH (17, 34). Structural isomers of each glucuronide were separated by HPLC on a reverse-phase column, and their structures were verified by mass spectrometry and NMR spectroscopy. Quantification of all vitamin D metabolites was performed by measuring the UV absorption at 264-nm wavelength and then calculated by using Beer’s law with the molar extinction coefficient of 18,300 mol/L. Escherichia coli-derived GUS (76,717 U/ml) in 50% glycerol solution was purchased from Sigma Aldrich and diluted in PBS before use.

Tissue Culture

Two adherent human adenocarcinoma colon cells, HT-29 (44-yr-old female Caucasian donor) and Caco2 (72-yr-old male Caucasian donor) from the American Type Culture Collection (Manassas, VA), were used to evaluate the response to 25OHD-Gluc. Cells were maintained at 37°C with 5% CO2 in DMEM medium (Gibco, Life Technologies, Grand Island, NY) for HT-29 cells or MEM medium (Corning Cellgro, Manassas, VA) for Caco2 cells. Maintenance media were supplemented with 0.2% penicillin-streptomycin and 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA). Cells were split at 80% confluency and maintained up to passage 20. Experiments were performed in 6-well plates, seeded with 2×106 cells in 3 ml maintenance medium. Twenty-four hours after being seeded, maintenance media were replaced with 3 ml low-serum media containing 0.1% FBS for treatment. The use of low-serum media is necessary to allow entry of 25OHD into cells because vitamin D-binding proteins in FBS prevent uptake of 25OHD and 25OHD-Gluc by most cells. Therefore, low-serum media more closely resembles the lack of intact serum proteins that would be observed in the lumen of the colon (33). Wells were treated with 100 nM 25OHD, 100 nM 25OHD-Gluc, or an equal volume of 100% EtOH vehicle (control) and cotreated with 3 units of E. coli-derived GUS in PBS or an equal volume of PBS vehicle. Each experimental plate contained one GUS-control and one PBS-control that were cotreated with EtOH vehicle and were used to calculate the relative expression from QPCR. The experiment was repeated, independently, three times (n = 3 per treatment). After a 16-h treatment incubation time, the media were replaced with 500 μl TRIzol Reagent (Invitrogen, Carlsbad, CA) to suspend and lyse cells, and then stored at −80°C for RNA extraction.

Animals

Male C57BL/6 mice were purchased from Charles River Laboratories (Kingston, NY). Mice were housed in solid-bottom ventilated cages maintained at constant temperatures (24–26°C) with a 12-h light-dark cycle. Standard rodent chow diet containing 1% calcium and 1.5 IU/g vitamin D3 on an as-fed basis (Teklad 2014, Harland Laboratories, Madison, WI) was offered ad libitum. All animal handling, treatment, and surgical procedures were approved by the Iowa State University Institutional Animal Care and Use Committee.

Animal Experiments

Oral dosing.

Oral dosing was used to evaluate first pass response by the intestine. Five-week-old mice were given a single bolus dose of 1 nmol 25OHD-Gluc, 25 pmol 1,25(OH)2D-Gluc (positive control), or an equal volume of EtOH (negative control) carried in 50 μl peanut oil. Each treatment group consisted of five animals (n = 5 mice per group). Mice were euthanized 4 h after gavage by inhalation of isoflurane followed by decapitation. Mice were dissected to remove a 3- to 5-cm section of proximal small intestine (duodenum) and of proximal colon. Dissected tissues were flushed with ice-cold 0.9% sodium chloride saline solution and cut into 1-cm segments for tissue analyses. The first segment of the intestinal tissue was removed and discarded. The second cm was collected into TRIzol Reagent, homogenized, and then stored at −80°C for RNA isolation and QPCR. In some experiments, a third segment was fixed in neutral-buffered formaldehyde for 24 h, and then transferred to 70% EtOH in RNase-free water for storage at room temperature.

Subcutaneous dosing.

Five-week-old mice were injected subcutaneously with 25OHD or 25OHD-Gluc at doses of 0.5, 1, and 2 nmol in 100 μl sterile propylene glycol. Control mice received subcutaneous injection of carrier alone. Four animals were treated per dose (n = 4 mice/dose). Mice were euthanized after 8 h. Blood was collected from the cervical stump into heparinized microfuge tubes and then centrifuged to separate plasma. Plasma 25OHD concentration was quantified by mass spectrometry (Heartland Assays, Ames, IA). Duodenal and colon tissues were dissected as described above for RNA isolation and QPCR analysis.

Surgical ligation.

Surgical ligation of the jejunum was used to prevent bile or digesta from reaching the colon as was done in Koszewski et al. (22). In intestinal ligation experiments, 9-wk-old mice were subjected to either surgical ligation of the jejunum or sham surgery before receiving 25OHD or 25OHD-Gluc delivered either orally or subcutaneously. Briefly, mice were anesthetized by inhalation of isoflurane and injected with 100 μl of 0.075% bupivacaine subcutaneously into muscle tissue on each sides of the surgical site of the abdomen for local anesthesia/ extended analgesia. A 2-cm midline incision was made into the abdomen. A section of jejunum was exteriorized, and a suture was placed around the section of intestine and tied tightly to block flow of digesta. Care was taken to place sutures to minimize interruption of mesenteric blood flow to the intestine. In some animals following ligation of the jejunum, the cecum also was exteriorized and 25OHD-Gluc in propylene glycol was injected into the lumen of the cecum (intracecum; IC) by using a 26-gauge needle. The intestine and cecum were returned to the peritoneal cavity, and then the abdomen and skin were closed in a single layer of suture. Sham surgical animals underwent the same anesthesia, midline incision, and exteriorized manipulation of the small intestine, but the segment of intestine was not ligated before being returned to the peritoneal cavity and the incision closed. After surgery, animals received either a subcutaneous injection (while still anesthetized) of 2 nmol of 25OHD or 25OHD-Gluc carried in 100 μl propylene glycol or an oral gavage (when mice were fully conscious and able to swallow at ~15 to 20 m postsurgery) of 2 nmol 25OHD, 25OHD-Gluc, or EtOH carried in 100 μl peanut oil. Each treatment group included 4–8 mice for each ligation and sham procedure. Eight hours after receiving their respective treatment, the mice were euthanized, as described above, and both the duodenum and colon were collected for QPCR analysis.

RNA Isolation

RNA was separated from cell and tissue extracts by TRIzol-chloroform separation and then passed through the RNeasy Mini Prep column (Qiagen, Germantown, MD) as previously described (32, 33). In short, chloroform was added to the TRIzol homogenate and centrifuged. The upper layer was collected, diluted in ethanol, and then transferred to the RNeasy column. The column was washed with buffers in accordance with manufacturer’s protocol, with an added sodium-potassium wash step. RNA was eluted by RNase-free water, quantified by UV absorbance at 260 nm and the 260/280 ratio, and then diluted to 0.5 µg RNA/μl. RNA (1 μg) was used as template for cDNA synthesis by Superscript III First Strand Synthesis (Invitrogen). cDNA was diluted with buffer (10 mM Tris and 1 mM EDTA in RNase-free water) to 60 μl for cell culture and 100 μl for tissue samples and then stored at −20°C.

QPCR

Messenger RNA was analyzed by QPCR with GAPDH and cytochrome P450 family 24 subfamily a1 (Cyp24) target probes for both human and mouse as previously described (32, 33). Briefly, Quanta SYBR Green (QuantaBio, Beverly, MA) was used to detect targets on a CFX96 C1000 thermocycler (Bio-Rad, Hercules, CA). Cyp24 was used as a measure of the VDR-mediated response as it has previously been shown to rapidly and robustly increase expression upon ligand binding and activation of VDR (20, 21). GAPDH was used to normalize expression of Cyp24. In cell culture, the respective experimental control was used to calculate relative expression, whereas in mice experiments the average normalized expression of control animals was used to calculate relative expression. Fold-change differences were calculated from 2ddCt, where ddCt = Relative Expression.

RNA In Situ Hybridization

Formalin-fixed, paraffin-embedded tissues were used for RNA in situ hybridization (RISH) of Cyp24 mRNA as previously described (32). Tissues were sectioned at 5 μm thickness and immediately hybridized with RNAscope 2.0 HD Red Manual Detection Kit (ACDbio, Newark, CA) by using probes for Cyp24 (sequence proprietary to ACDbio) and counterstained with hematoxylin. Images were obtained by a light microscopy at ×20 magnification, and mRNA was detected in red chromogenic dye.

Statistical Analyses

Statistical analyses were performed by GraphPad Prism 5 (GraphPad Software, San Diego, CA). Analyses include t tests, 1-way ANOVA comparisons with Tukey adjustments for pairwise comparison and 2-way ANOVA comparisons with Bonferroni Adjustments as indicated. Data are presented as means ± SE. Statistical significance was notated when P < 0.05.

RESULTS

In Vitro Response to 25OHD and 25OHD-Gluc

Both HT-29 and Caco2 cell lines demonstrated robust VDR-mediated responses when incubated with 100 nM 25OHD in low-serum conditions (Fig. 1). The response to 25OHD was not affected by addition of GUS to the media. Incubation with equimolar 25OHD-Gluc produced only a small increase in Cyp24 mRNA that was significantly different from controls in Caco2 cells (P < 0.0001) but not present in HT29 cells (P = 0.1). Cotreatment of 25OHD-Gluc with GUS enabled robust responses that were 5 Ct (32-fold) greater than without GUS for both cell lines. Based on these results, it is reasonable to consider that the cells and media may have limited intrinsic GUS activity.

Fig. 1.

Fig. 1.

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Bacterial glucuronidase (GUS) aids in the 25-hydroxyvitamin D3-3β-glucuronic acid (25OHD-Gluc)-mediated response in human HT-29 (A) and Caco2 (B) colon adenocarcinoma cell lines. Cells were incubated for 16 h in media treated with 100 nM 25-hydroxyvitamin D (25OHD) or 25OHD-Gluc and cotreated with 1 U/ml Escherichia coli GUS or PBS in low-serum media conditions. Relative expression values are QPCR Ct units normalized to GAPDH and relative to respective experimental EtOH-treated controls. Triangles represent each data point, n = 3 per treatment, bars are means ± SE, #P < 0.001, t test with EtOH-treated cells with respective Control or GUS media. *P < 0.001, 2-way ANOVA Bonferroni posttest.

Intestinal Responses to Oral Administration of Vitamin D Glucuronides in Mice

Oral administration of 1 nmol 25OHD-Gluc or 25 pmol 1,25(OH)2D-Gluc had no effect on Cyp24 mRNA expression in the duodenum after 4 h (Fig. 2A). However, both 1 nmol 25OHD-Gluc and 25 pmol 1,25(OH)2D-Gluc induced Cyp24 mRNA in the colon (Fig. 2B). Localization of Cyp24 mRNA in the glucuronide-treated colons by RISH demonstrates that the responses are identical between the two glucuronide substrates and appear in the epithelial cells closest to the lumen, but not the submucosal, muscularis, or lymph regions (Fig. 2C). No Cyp24 mRNA was observed in deeper regions of the colon crypts by either vitamin D compound.

Fig. 2.

Fig. 2.

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Oral administration of 25-hydroxyvitamin D3-3β-glucuronic acid (25OHD-Gluc) and 1,25-dihydroxyvitamin D3-25- glucuronic acid (1,25(OH)2D-Gluc) in mice results in upregulation of Cyp24 mRNA in the colon but not the duodenum. Mice were given vehicle, 1 nmol of 25OHD-Gluc, or 0.25 nmol 1,25(OH)2D-Gluc, and the duodenum (A) and colon (B) were harvested 4 h later. Cyp24 mRNA relative expression values are QPCR Ct units normalized to GAPDH and relative to the mean of control animal data. Triangles represent each data point, bars are means ± SE, n = 5 mice/group. *P < 0.05, ANOVA Tukey’s pairwise comparisons of relative expression values. Representative images of colon cross-sections with RNA in situ hybridization (RISH) for Cyp24 mRNA (C) as indicated by red stain as demonstrated by arrows with hematoxylin counterstain. Scale bars indicate 100 µm at ×20 magnification.

Intestinal Responses to Subcutaneous Administration of 25OHD and 25OHD-Gluc in Mice

When given subcutaneously, 2 nmol 25OHD increased plasma 25OHD and elicited a response in the duodenum 8 h after injection with no effect on the colon (Fig. 3). Lesser doses of 25OHD (1 nmol and 0.5 nmol) also increased plasma 25OHD concentrations but had no effect on expression of duodenal or colon Cyp24 mRNA 8 h after injection. Conversely, both 1- and 2-nmol doses of 25OHD-Gluc elicited significant increases in the Cyp24 mRNA response in the colon (P = 0.04 and P = 0.02, respectively), yet only the 2-nmol dose of 25OHD-Gluc raised serum 25OHD significantly. Subcutaneous 25OHD-Gluc injection had no effect on Cyp24 mRNA in the duodenum.

Fig. 3.

Fig. 3.

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Effect of subcutaneous 25-hydroxyvitamin D (25OHD) and 25-hydroxyvitamin D3-3β-glucuronic acid (25OHD-Gluc) on plasma 25OHD concentration (A), duodenal Cyp24 mRNA (B), and colon Cyp24 mRNA (C). Mice were treated with 0 (control), 0.5, 1, or 2 nM of either 25OHD or 25OHD-Gluc, and tissues were harvested 8 h later. Cyp24 mRNA relative expression values are QPCR Ct units normalized to GAPDH and calculated relative to the mean of control data. Triangles represent each data point, bars are means ± SE, n = 4 mice/group. *P < 0.05, by ANOVA with Tukey-Kramer pairwise comparisons analyzed per metabolite.

Effects of Intestinal Ligation on Responses to 25OHD

Surgical ligation of the small intestine physically blocks passage of digesta and bile to the distal intestine. The ligation procedure itself caused no changes in Cyp24 expression compared with the sham procedure in both the duodenum and colon (Figs. 4A and 5A). Oral administration of 25OHD induced Cyp24 mRNA compared with control treatment in the duodenum following both the sham (P = 0.04) and the ligation procedures (P = 0.04) and was not affected by surgical procedure (P = 0.4; Fig. 4B). Subcutaneous administration of 25OHD did not induce Cyp24 mRNA in the duodenum (Fig. 4B) despite demonstrating a significant response in nonsurgical conditions (Fig. 3B), suggesting that anesthesia may have interfered with bile excretion. The colon exhibited an increase in Cyp24 mRNA from oral 25OHD following the sham surgery (P = 0.01) but not following the ligation surgery (P > 0.05; Fig. 5B). These results suggest that the oral 25OHD moved to the colon via intestinal passage and was blocked by the surgical ligation. The complete lack of responses to subcutaneous 25OHD in the duodenum and colon from either ligation or sham surgery suggests that circulating 25OHD (which is largely bound to vitamin D-binding proteins and albumin) is not able to stimulate these tissues, perhaps due to an inability to cross the basolateral membrane of the intestines.

Fig. 4.

Fig. 4.

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Duodenal responses to 25-hydroxyvitamin D (25OHD) and 25-hydroxyvitamin D3-3β-glucuronic acid (25OHD-Gluc) following jejunal ligation surgery that blocks flow of digesta to the distal intestine. Mice were subjected to either sham or ligation surgery and then received vehicle (Control, A), 2 nmol 25OHD (B), or 2 nmol 25OHD-Gluc (C), via oral (Oral), subcutaneous (SubQ), or intracecum (IC) routes. Relative expression values are QPCR Ct units normalized to GAPDH and calculated relative to the mean of control data. Triangles represent each data point, bars are means ± SE, n = 5–8 mice/group. *P < 0.05, t test comparison with respective control or as indicated.

Fig. 5.

Fig. 5.

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Colonic responses to 25-hydroxyvitamin D (25OHD) and 25-hydroxyvitamin D3-3β-glucuronic acid (25OHD-Gluc) following intestinal ligation to block digesta from entering into the colon. Mice were subjected to either sham or ligation surgery and then received vehicle (Control, A), 2 nmol 25OHD (B), or 2 nmol 25OHD-Gluc (C), via oral (Oral), subcutaneous (SubQ), or intracecum (IC) routes. Relative expression values are QPCR Ct units normalized to GAPDH and calculated relative to the mean of control data. Triangles represent each data point, bars are means ± SE, n = 5–8 mice/group. *P < 0.05, t test comparison with respective control or as indicated.

Effects of Intestinal Ligation on Responses to 25OHD-Gluc

As expected, 25OHD-Gluc treatment had no effect on the expression of Cyp24 at the duodenum (above the surgical site) in both surgical conditions and by both routes of administration (Fig. 4C). In the colon, however, orally administered 25OHD-Gluc successfully increased Cyp24 expression following the sham surgery (P = 0.01, Fig. 5C). The response to oral 25OHD-Gluc was abolished in surgically ligated mice (P = 0.007 between sham and ligated groups; Fig. 5C). Subcutaneous administration of 25OHD-Gluc in sham surgery mice tended to increase Cyp24 expression in the colon from that of the control animals, but this difference was not statistically significant (P = 0.09). Again, colon Cyp24 expression was impaired in surgically ligated mice treated with subcutaneous 25OHD-Gluc (P = 0.049 between sham and ligated groups; Fig. 5C). To ensure that the colon Cyp24 response to 25OHD-Gluc was not impaired by the ligation surgery, we also injected 25OHD-Gluc directly into the cecum (IC) at the time of the ligation and euthanized these mice 4–8 h later. Colons of mice that received 25OHD-Gluc IC (below the ligated intestine) had greatly increased Cyp24 expression in the colon (P = 0.02). This IC injection verified the ability of colon bacteria to cleave the glucuronide and for colon epithelial cells to respond to a VDR ligand was intact despite ligation surgery.

DISCUSSION

The present study has shown that 25OHD-Gluc stimulates the VDR-mediated pathway in the colon of mice by both oral and subcutaneous routes of administration. It is evident that vitamin D metabolites, such as 25OHD and 1,25(OH)2D, are removed from circulation and glucuronidated by the liver for excretion into bile (17, 40). As the glucuronide-containing bile passes through the intestinal tract, it encounters gut bacteria with ample GUS enzyme activity to cleave beta-linked glucuronic acids (16, 30, 37). Our use of a purified bacterial GUS enzyme in cell culture assays confirmed that 25OHD-Gluc must have the glucuronide cleaved from the 25OHD to effectively induce vitamin D-dependent gene expression in cells. These results extend our previous study showing that colon contents have the capacity to cleave the glucuronic acid moiety on 1,25(OH)2D-Gluc to yield measurable quantities of liberated 1,25(OH)2D (17). Our in vitro results also indicate that the conjugated 25OHD-Gluc would have minimal biological activity until reaching the terminal ileum, cecum, or colon where GUS is abundant (16, 37). Indeed, mice that are given 25OHD-Gluc elicited responses only in the colon where elevated GUS activity is present.

Colon epithelial cells are the primary target cells for 25OHD-Gluc. By using RNAscope to detect Cyp24 mRNA in cross-sections of the colon, we found that only colonic epithelial cells close to the lumen exhibited the Cyp24 response. Cells deeper within the crypts did not exhibit a response even though previous studies have shown VDR to be expressed throughout the epithelium (4, 6, 15, 35, 39). These data are similar to our previous findings that 1,25(OH)2D-Gluc also affects colon epithelial cells that are located closest to the lumen and that the same distribution of the response occurs in the duodenum of mice when treated with unconjugated metabolites, 1,25(OH)2D and 25OHD (32, 33). Interestingly, a recent study published by Fleet and Reyes-Fernandez (10) also suggested that increases in VDR content in a tissue may not necessarily equate to an increase in vitamin D response. Based on our accumulating data, we believe that 25OHD enters colon epithelial cells via the apical membrane to stimulate the Cyp24 mRNA response. If, instead, the cells received stimulation from blood-borne vitamin D metabolites (25OHD or 1,25(OH)2D) at the basolateral membrane, then cells within crypt regions (that also express VDR) should have exhibited similar responses to cells closer to the lumen. Furthermore, raising blood 25OHD concentrations by subcutaneous injection of 25OHD should hypothetically induce a large response throughout the entire intestine, but it did not. We also found that responses to 25OHD-Gluc, which has been shown to be produced from 25OHD by the liver and excreted in the bile (13, 40), occurred in the colon only when 25OHD could be liberated in the lumen following enzymatic cleavage of 25OHD-Gluc by gut GUS. Taken together, it is apparent that apical entry of vitamin D metabolites is of great importance for the colon epithelial cells and may apply in additional regions of the intestine, although more testing would be needed to confirm this action elsewhere along the intestinal tract.

When 25OHD was administered subcutaneously it caused increases in plasma 25OHD concentration, but colon and duodenal responses were absent, except in the duodena of mice that received the largest dose of subcutaneous 25OHD (2 nmol). Recently, we had studied the effect of oral 25OHD on the mouse duodenum and determined that 0.2 nmol 25OHD was needed to induce a response (33). We showed that when inside the intestine, 25OHD acts as a VDR agonist and that this 0.2-nmol dose of 25OHD was estimated to be at the upper end of a potential dietary intake, yet still physiologically reasonable and unlikely to cause hypercalcemia. If basolateral absorption was the only way for vitamin D signaling to stimulate the duodenum, then why would a subcutaneous dose require 10 times more 25OHD than an oral dose? The discrepancy between routes of administration can be justified two ways: 1) much of the subcutaneous dose immediately became bound to serum proteins and could not enter the duodenal cells by crossing the basolateral membrane, or 2) a portion of the subcutaneous dose was excreted via bile to elicit the agonistic hormonal response at the apical membrane in the duodenum. Additional research is necessary to discern these differences and firmly decipher the route of entry for active vitamin D metabolites in the duodenum. As for the colon, a study conducted in mice and rats demonstrated that a bolus dose of 1,25(OH)2D, administered orally or subcutaneously, also has no effect on colon gene expression (22). If hormonal stimulation did occur via basolateral membrane, then we would expect a direct and consistent correlation between plasma hormone concentrations (i.e., 1,25(OH)2D or free-25OHD) and gene expression. This was not observed within the colon in the present study or our previous studies; thus, we argue that vitamin D metabolites do not cross the basolateral membrane of the colon epithelium to a significant degree and that hormonal stimulation of the colon epithelium occurs across the apical membrane.

To provide further proof that the colon responds specifically to intra-luminal 25OHD after GUS cleavage, we performed surgical ligations that block the passage of bile and digesta. In mice that underwent sham surgery with patent intestinal tract, 2 nmol 25OHD-Gluc upregulated Cyp24 expression in the colon. The response was not as robust as was observed in acutely dosed mice without surgical intervention. We suspect that the sham surgery and anesthesia slowed gut motility enough to decrease the amount of 25OHD-Gluc that reached the colon during the 8-h study. In mice that underwent ligation surgery to block entry of bile and digesta into the colon, the Cyp24 response to 25OHD-Gluc was absent regardless of route of administration. These results coincide with our previous findings that ligation surgery in mice and rats also interferes with colon responses to oral and subcutaneous 1,25(OH)2D-Gluc (22). Taken together, we believe these data prove that the apical membrane of the colon epithelium is the major avenue used by 25OHD and 1,25(OH)2D to stimulate vitamin D-dependent gene expression.

25OHD-Gluc is naturally present in bile at higher concentrations than 1,25(OH)2D-Gluc, and, therefore, may be the primary and perhaps only means of providing vitamin D stimulation for the colon. The enterohepatic circulation for vitamin D metabolites, such as glucuronides, had been proposed by Kumar and colleagues decades ago to be physiologically relevant (1, 42). Unfortunately, shortly thereafter these conjugated metabolites were considered to lack biological function and were ascribed a role as excretory metabolites in a pathway for removing excessive amounts of vitamin D metabolites from the body (8, 11, 14, 23, 29). Those studies, however, focused on the calcemic activities within the duodenum and ignored the role that these compounds might play in the colon. A model proposed by Gao et al. (13) describes enterohepatic transport for the glucuronide conjugate of 25OHD that assumes liberated 25OHD needs to be activated by enteric 1α-hydroxylase to provide 1,25(OH)2D for local action in the colon. A recent study by our laboratory, however, had demonstrated that 25OHD, when present within the intestinal lumen, acts as a hormonal agonist on the duodenum of mice and was not being converted to 1,25(OH)2D by epithelial cells (33). Because of the agonistic action of 25OHD on the apical membrane of the intestinal epithelium, we propose a revised model of enterohepatic signaling that is independent from local 1,25(OH)2D production. In this model, calcium absorption in the distal intestine (cecum and colon) is dependent on 25OHD-Gluc and 1,25(OH)2D-Gluc. A portion of circulating 25OHD and 1,25(OH)2D is conjugated to a glucuronide by hepatocytes and secreted within the bile. Upon reaching the large intestine, bacterial GUS cleaves the glucuronide, thus liberating 25OHD and 1,25(OH)2D within the lumen. The 25OHD and 1,25(OH)2D move into the epithelial cells across the apical membrane to bind to VDR and initiate production of vitamin D-dependent proteins, including those involved in calcium absorption. Indeed, active calcium transport by the colon has been recognized to occur in a vitamin D-dependent manner (6, 18, 19, 26, 41). Knockout and transgenic mouse studies that manipulate the expression of VDR in the distal intestines provide additional support for the role of the colon in intestinal calcium absorption (7, 9, 31). Our model of the glucuronide conjugate carrying 25OHD to the colon can be applied to other known conjugates such as the glycoside and the sulfate. Interestingly, the 25OHD-sulfate is present in blood and bile in much greater concentrations, and, depending on the sulfatase activity of the gut, may be a valuable component of enterohepatic vitamin D signaling (12, 13, 20, 43).

Our in vivo studies were performed on vitamin D-sufficient mice that consume a diet that meets the Association of American Feed Control Officials standards for vitamin D and calcium content in rodent diets. Humans, particularly in the United States, tend to have lower circulating 25OHD concentrations and consume a recommended diet that is proportionally lower in calcium and vitamin D than the minimal standards for rodent diets. This may lead to insufficient excretion of 25OHD-Gluc in the bile and insufficient vitamin D influence on colon epithelium functions. Our data focused only on the expression of Cyp24 as an indicator of the VDR-mediated response, as these were necessarily acute studies involving surgically ligated intestines. Additional vitamin D-responsive genes, such as TRPV6, PMCA1, and Calbindin D9k, are directly involved in calcium uptake, but require more time to reach peak expression in the colon (22). The extended time frame in surgical ligation studies would not be reasonable or ethical, as occlusion of the digestive tract causes accumulation of secreted fluid orad to the ligature which can cause pain in the animals. Nonsurgical in vivo studies would also be complicated because administration of 25OHD or 25OHD-Gluc increases plasma 25OHD concentrations in a matter of hours. A portion of the circulating 25OHD would then be glucuronidated and sent into bile. Therefore, we would not be able to differentiate the effects of 25OHD-Gluc treatment from those of increased plasma 25OHD that eventually passed in the bile in a hypothetical study that was long enough to see upregulation of colon CaBP. A recent study by Jiang and colleagues demonstrated mRNA induction for Trpv6 and Calbindin D9k as well as Cyp24 in the duodenum and colon of mice in response to similarly modified 1,25(OH)2D compounds (20). Future studies should focus on genes involved in calcium transport, calcium absorption, as well as aspects pertaining to epithelial health and immune function to fully understand the impact of 25OHD-Gluc actions in the colon.

In conclusion, we identified the colon as a physiological target of 25OHD-Gluc. Our data demonstrate that 25OHD-Gluc is transported to the colon via bile or digesta where it is cleaved by bacterial enzymes into free 25OHD. The free 25OHD crosses the apical membrane of the cells and acts as a VDR agonist. This mechanisms of enterohepatic signaling and of VDR agonist action may be important factors in stimulating calcium absorption in the colon and maintaining colon cell health.

GRANTS

This work was supported, in part, by National Institutes of Health Grant 2R15CA173628-02.

DISCLOSURES

J.P.G and R.L.H jointly own a company (Glycomyr, Inc, Ames, Iowa) that synthesizes vitamin D glucuronides. All other authors do not have disclosures to report.

AUTHOR CONTRIBUTIONS

C.J.R., N.J.K., and J.P.G. conceived and designed research; C.J.R. and N.J.K. performed experiments; C.J.R. and R.L.H. analyzed data; C.J.R., N.J.K., R.L.H., D.C.B., and J.P.G. interpreted results of experiments; C.J.R. prepared figures; C.J.R. drafted manuscript; C.J.R., N.J.K., D.C.B., and J.P.G. edited and revised manuscript; C.J.R., N.J.K., D.C.B., and J.P.G. approved final version of manuscript.

ACKNOWLEDGMENTS

The authors thank C. Martens for invaluable technical assistance throughout this study.

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