Implication of intestinal VDR deficiency in inflammatory bowel disease

Background

To investigate the function of the intestinal Vdr gene in inflammatory bowel disease (IBD), in conjunction with the discovery of possible metabolic markers for IBD using intestine-specific Vdr knockout mice.

Methods

Vdr^ΔIEpC mice were generated, phenotyped and treated with a time-course of 3% dextran sulfate sodium (DSS) to induce colitis. Colitis was diagnosed by evaluating clinical symptoms and intestinal histopathology. Gene expression analysis was carried out. In addition, metabolic markers of IBD were explored by metabolomics.

Results

Vdr^ΔIEpC mice showed abnormal body size, colon structures and feces color. Calcium, collagen, and intestinal proliferation-related gene expression were all decreased, and serum alkaline phosphatase was highly increased. In the acute model which was treated with 3% DSS for six days, Vdr^ΔIEpC mice showed a high score of IBD symptoms; enlarged mucosal layer and damaged muscularis layer. In the recovery experiment model, where mice were treated with 3% DSS for four days and water for three days, Vdr^ΔIEpC mice showed a high score of IBD symptoms; severe damage of mucosal layer and increased expression of genes encoding proinflammatory cytokines. Feces metabolomics revealed decreased concentrations of taurine, taurocholic acid, taurodeoxycholic acid and cholic acid in Vdr^ΔIEpC mice.

Conclusions

Disruption of the intestinal Vdr gene showed phenotypical changes that may exacerbate IBD. These results suggest that VDR may play an important role in IBD.General significanceVDR function has been implicated in IBD. This is of value for understanding the etiology of IBD and for development of diagnostic biomarkers for IBD.     

TGF-β-SMAD3 signaling mediates hepatic bile acid and phospholipid metabolism following lithocholic acid-induced liver injury

Transforming growth factor-β (TGFβ) is activated as a result of liver injury, such as cholestasis. However, its influence on endogenous metabolism is not known. This study demonstrated that TGFβ regulates hepatic phospholipid and bile acid homeostasis through MAD homolog 3 (SMAD3) activation as revealed by lithocholic acid-induced experimental intrahepatic cholestasis. Lithocholic acid (LCA) induced expression of TGFB1 and the receptors TGFBR1 and TGFBR2 in the liver. In addition, immunohistochemistry revealed higher TGFβ expression around the portal vein after LCA exposure and diminished SMAD3 phosphorylation in hepatocytes from Smad3-null mice. Serum metabolomics indicated increased bile acids and decreased lysophosphatidylcholine (LPC) after LCA exposure. Interestingly, in Smad3-null mice, the metabolic alteration was attenuated. LCA-induced lysophosphatidylcholine acyltransferase 4 (LPCAT4) and organic solute transporter β (OSTβ) expression were markedly decreased in Smad3-null mice, whereas TGFβ induced LPCAT4 and OSTβ expression in primary mouse hepatocytes. In addition, introduction of SMAD3 enhanced the TGFβ-induced LPCAT4 and OSTβ expression in the human hepatocellular carcinoma cell line HepG2. In conclusion, considering that Smad3-null mice showed attenuated serum ALP activity, a diagnostic indicator of cholangiocyte injury, these results strongly support the view that TGFβ-SMAD3 signaling mediates an alteration in phospholipid and bile acid metabolism following hepatic inflammation with the biliary injury.     

Mechanisms of Enhancer-mediated Hormonal Control of Vitamin D Receptor Gene Expression in Target Cells

The biological actions of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) are mediated by the vitamin D receptor (VDR), whose expression in bone cells is regulated positively by 1,25(OH)₂D₃, retinoic acid, and parathyroid hormone through both intergenic and intronic enhancers. In this report, we used ChIP-sequencing analysis to confirm the presence of these Vdr gene enhancers in mesenchyme-derived bone cells and to describe the epigenetic histone landscape that spans the Vdr locus. Using bacterial artificial chromosome-minigene stable cell lines, CRISPR/Cas9 enhancer-deleted daughter cell lines, transient transfection/mutagenesis analyses, and transgenic mice, we confirmed the functionality of these bone cell enhancers in vivo as well as in vitro. We also identified VDR-binding sites across the Vdr gene locus in kidney and intestine using ChIP-sequencing analysis, revealing that only one of the bone cell-type enhancers bound VDR in kidney tissue, and none were occupied by the VDR in the intestine, consistent with weak or absent regulation by the 1,25(OH)₂D₃ hormone in these tissues, respectively. However, a number of additional sites of VDR binding unique to either kidney or intestine were present further upstream of the Vdr gene, suggesting the potential for alternative regulatory loci. Importantly, virtually all of these regions retained histone signatures consistent with those of enhancers and exhibited unique DNase I hypersensitivity profiles that reflected the potential for chromatin access. These studies define mechanisms associated with hormonal regulation of the Vdr and hint at the differential nature of VDR binding activity at the Vdr gene in different primary target tissues in vivo.     

Vitamin D Receptor Gene Ablation in the Conceptus Has Limited Effects on Placental Morphology,Function and Pregnancy Outcome

Vitamin D deficiency has been implicated in the pathogenesis of several pregnancy complications attributed to impaired or abnormal placental function, but there are few clues indicating the mechanistic role of vitamin D in their pathogenesis. To further understand the role of vitamin D receptor (VDR)-mediated activity in placental function, we used heterozygous Vdr ablated C57Bl6 mice to assess fetal growth, morphological parameters and global gene expression in Vdr null placentae. Twelve Vdr ^+/- dams were mated at 10–12 weeks of age with Vdr ^+/- males. At day 18.5 of the 19.5 day gestation in our colony, females were euthanised and placental and fetal samples were collected, weighed and subsequently genotyped as either Vdr ^+/+, Vdr ^+/- or Vdr ^-/-. Morphological assessment of placentae using immunohistochemistry was performed and RNA was extracted and subject to microarray analysis. This revealed 25 genes that were significantly differentially expressed between Vdr ^+/+ and Vdr ^-/- placentae. The greatest difference was a 6.47-fold change in expression of Cyp24a1 which was significantly lower in the Vdr ^-/- placentae (P<0.01). Other differentially expressed genes in Vdr ^-/- placentae included those involved in RNA modification (Snord123), autophagy (Atg4b), cytoskeletal modification (Shroom4), cell signalling (Plscr1, Pex5) and mammalian target of rapamycin (mTOR) signalling (Deptor and Prr5). Interrogation of the upstream sequence of differentially expressed genes identified that many contain putative vitamin D receptor elements (VDREs). Despite the gene expression differences, this did not contribute to any differences in overall placental morphology, nor was function affected as there was no difference in fetal growth as determined by fetal weight near term. Given our dams still expressed a functional VDR gene, our results suggest that cross-talk between the maternal decidua and the placenta, as well as maternal vitamin D status, may be more important in determining pregnancy outcome than conceptus expression of VDR.     

Lithocholic acid derivatives act as selective vitamin D receptor modulators without inducing hypercalcemia

1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a vitamin D receptor (VDR) ligand, regulates calcium homeostasis and also exhibits noncalcemic actions on immunity and cell differentiation. In addition to disorders of bone and calcium metabolism, VDR ligands are potential therapeutic agents in the treatment of immune disorders, microbial infections, and malignancies. Hypercalcemia, the major adverse effect of vitamin D(3) derivatives, limits their clinical application. The secondary bile acid lithocholic acid (LCA) is an additional physiological ligand for VDR, and its synthetic derivative, LCA acetate, is a potent VDR agonist. In this study, we found that an additional derivative, LCA propionate, is a more selective VDR activator than LCA acetate. LCA acetate and LCA propionate induced the expression of the calcium channel transient receptor potential vanilloid type 6 (TRPV6) as effectively as that of 1alpha,25-dihydroxyvitamin D(3) 24-hydroxylase (CYP24A1), whereas 1,25(OH)(2)D(3) was more effective on TRPV6 than on CYP24A1 in intestinal cells. In vivo experiments showed that LCA acetate and LCA propionate effectively induced tissue VDR activation without causing hypercalcemia. These bile acid derivatives have the ability to function as selective VDR modulators.     

Promoter-, cell-, and ligand-specific transactivation responses of the VDRB1 isoform

The vitamin D receptor (VDR) mediates the effects of 1,25(OH)(2)D(3), the active form of vitamin D. The human VDRB1 isoform differs from the originally described VDR by an N-terminal extension of 50 amino acids. Here we investigate cell-, promoter-, and ligand-specific transactivation by the VDRB1 isoform. Transactivation by these isoforms of the cytochrome P450 CYP24 promoter was compared in kidney (HEK293 and COS1), tumor-derived colon (Caco-2, LS174T, and HCT15), and mammary (HS578T and MCF7) cell lines. VDRB1 transactivation in response to 1,25(OH)(2)D(3) was greater in COS1 and HCT15 cells (145%), lower in HEK293 and Caco-2 cells (70-85%) and similar in other cell lines tested. By contrast, on the cytochrome P450 CYP3A4 promoter, 1,25(OH)(2)D(3)-induced VDRB1 transactivation was significantly lower than VDRA in Caco-2 (68%), but comparable to VDRA in HEK293 and COS1 cells. Ligand-dependence of VDRB1 differential transactivation was investigated using the secondary bile acid lithocholic acid (LCA). On the CYP24 promoter LCA-induced transactivation was similar for both isoforms in COS1, whereas in Caco-2 and HEK293 cells VDRB1 was less active. On the CYP3A4 promoter, LCA activation of VDRB1 was comparable to VDRA in all the cell lines tested. Mutational analysis indicated that both the 1,25(OH)(2)D(3) and LCA-regulated activities of both VDR isoforms required a functional ligand-dependent activation function (AF-2) domain. In gel shift assays VDR:DNA complex formation was stronger in the presence of 1,25(OH)(2)D(3) than with LCA. These results indicate that regulation of VDRB1 transactivation activity is dependent on cellular context, promoter, and the nature of the ligand.     

Mice with Chimeric Livers Are an Improved Model for Human Lipoprotein Metabolism

Objective

Rodents are poor model for human hyperlipidemias because total cholesterol and low density lipoprotein levels are very low on a normal diet. Lipoprotein metabolism is primarily regulated by hepatocytes and we therefore assessed whether chimeric mice extensively repopulated with human cells can model human lipid and bile acid metabolism.

Design

FRG [ F ah(−/−) R ag2(−/−)Il2r g (−/−)]) mice were repopulated with primary human hepatocytes. Serum lipoprotein lipid composition and distribution (VLDL, LDL, and HDL) was analyzed by size exclusion chromatography. Bile was analyzed by LC-MS or by GC-MS. RNA expression levels were measured by quantitative RT-PCR.

Results

Chimeric mice displayed increased LDL and VLDL fractions and a lower HDL fraction compared to wild type, thus significantly shifting the ratio of LDL/HDL towards a human profile. Bile acid analysis revealed a human-like pattern with high amounts of cholic acid and deoxycholic acid (DCA). Control mice had only taurine-conjugated bile acids as expcted, but highly repopulated mice had glycine-conjugated cholic acid as found in human bile. RNA levels of human genes involved in bile acid synthesis including CYP7A1, and CYP27A1 were significantly upregulated as compared to human control liver. However, administration of recombinant hFGF19 restored human CYP7A1 levels to normal.

Conclusion

Humanized-liver mice showed a typical human lipoprotein profile with LDL as the predominant lipoprotein fraction even on a normal diet. The bile acid profile confirmed presence of an intact enterohepatic circulation. Although bile acid synthesis was deregulated in this model, this could be fully normalized by FGF19 administration. Taken together these data indicate that chimeric FRG-mice are a useful new model for human lipoprotein and bile-acid metabolism.     

Impairment of Bilirubin Clearance and Intestinal Interleukin-6 Expression in Bile Duct-Ligated Vitamin D Receptor Null Mice

The vitamin D receptor (VDR) mediates the physiological and pharmacological actions of 1α,25-dihydroxyvitamin D₃ in bone and calcium metabolism, cellular growth and differentiation, and immunity. VDR also responds to secondary bile acids and belongs to the NR1I subfamily of the nuclear receptor superfamily, which regulates expression of xenobiotic metabolism genes. When compared to knockout mouse investigations of the other NR1I nuclear receptors, pregnane X receptor and constitutive androstane receptor, an understanding of the role of VDR in xenobiotic metabolism remains limited. We examined the effect of VDR deletion in a mouse model of cholestasis. We performed bile duct ligation (BDL) on VDR-null mice and compared blood biochemistry, mRNA expression of genes involved in bile acid and bilirubin metabolism, cytokine production, and expression of inflammatory regulators with those of wild-type mice. VDR-null mice had elevated plasma conjugated bilirubin levels three days after BDL compared with wild-type mice. Urine bilirubin levels and renal mRNA and/or protein expression of multidrug resistance-associated proteins 2 and 4 were decreased in VDR-null mice, suggesting impaired excretion of conjugated bilirubin into urine. While VDR-null kidney showed mRNA expression of interleukin-6 (IL-6) after BDL and VDR-null macrophages had higher IL-6 protein levels after lipopolysaccharide stimulation, the induction of intestinal Il6 mRNA expression and plasma IL-6 protein levels after BDL was impaired in VDR-null mice. Immunoblotting analysis showed that expression of an immune regulator, IκBα, was elevated in the jejunum of VDR-null mice, a possible mechanism for the attenuated induction of Il6 expression in the intestine after BDL. Increased expression of IκBα may be a consequence of compensatory mechanisms for VDR deletion. These results reveal a role of VDR in bilirubin clearance during cholestasis. VDR is also suggested to contribute to tissue-selective immune regulation.     

Lithocholic acid down-regulation of NF-kappaB activity through vitamin D receptor in colonic cancer cells

Lithocholic acid (LCA), a secondary bile acid, is a vitamin D receptor (VDR) ligand. 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the hormonal form of vitamin D, is involved in the anti-inflammatory action through VDR. Therefore, we hypothesize that LCA acts like 1,25(OH)(2)D(3) to drive anti-inflammatory signals. In present study, we used human colonic cancer cells to assess the role of LCA in regulation of the pro-inflammatory NF-kappaB pathway. We found that LCA treatment increased VDR levels, mimicking the effect of 1,25(OH)(2)D(3). LCA pretreatment inhibited the IL-1beta-induced IkappaBalpha degradation and decreased the NF-kappaB p65 phosphorylation. We also measured the production of IL-8, a well-known NF-kappaB target gene, as a read-out of the biological effect of LCA expression on NF-kappaB pathway. LCA significantly decreased IL-8 secretion induced by IL-1beta. These LCA-induced effects were very similar to those of 1,25(OH)(2)D(3.) Thus, LCA recapitulated the effects of 1,25(OH)(2)D(3) on IL-1beta stimulated cells. Mouse embryonic fibroblast (MEF) cells lacking VDR have intrinsically high NF-kappaB activity. LCA pretreatment was not able to prevent TNFalpha-induced IkappaBalpha degradation in MEF VDR (-/-), whereas LCA stabilized IkappaBalpha in MEF VDR (+/-) cells. Collectively, our data indicated that LCA activated the VDR to block inflammatory signals in colon cells.     

Maternal Vitamin D Status in Preeclampsia: Seasonal Changes Are Not Influenced by Placental Gene Expression of Vitamin D Metabolizing Enzymes

Preeclampsia, a hypertensive disorder in pregnancy develops in 2–8% of pregnancies worldwide. Winter season and vitamin D deficiency have been associated with its onset.

Objective

To investigate the influence of season on maternal vitamin D status and placental vitamin D metabolism.

Methods

25-OH vitamin D and 1,25-(OH)₂ vitamin D were measured in maternal serum obtained during the winter or summer months from 63 pregnant women at delivery (43 healthy, 20 preeclampsia). In a subgroup, mRNA expression of CYP24A1 (24-hydroxylase), CYP27B1 (1α-hydroxylase) and VDR (vitamin D receptor) were quantified by real time PCR in placental samples of 14 women with normal pregnancies and 13 with preeclampsia.

Results

In patients with preeclampsia,25-OH vitamin D levels were lower, but differed significantly from controls only in summer (18.21±17.1 vs 49.2±29.2 ng/mL, P<0.001), whereas 1,25-(OH)₂ vitamin D levels were significantly lower only in winter (291±217 vs 612.3±455 pmol/mL, P<0.05). A two-factorial analysis of variance produced a statistically significant model (P<0.0001) with an effect of season (P<0.01) and preeclampsia (P = 0.01) on maternal 25-OH vitamin D levels, as well as a significant interaction between the two variables (P = 0.02). Placental gene expression of CYP24A1, CYP27B1, and VDR did not differ between groups or seasons. A negative correlation between placental gene expression of CYP24A1 and CYP27B1 was observed only in healthy controls (r = −0.81, P<0.0001).

Summary

Patients with preeclampsia displayed lower vitamin D serum levels in response to seasonal changes.The regulation of placental CYP24A1, but not of the VDR or CYP27B1 might be altered in preeclampsia.     

Role of Vitamin D3 in Modulation of ΔNp63α Expression during UVB Induced Tumor Formation in SKH-1 Mice

ΔNp63α, a proto-oncogene, is up-regulated in non-melanoma skin cancers and directly regulates the expression of both Vitamin D receptor (VDR) and phosphatase and tensin homologue deleted on chromosome ten (PTEN). Since ΔNp63α has been shown to inhibit cell invasion via regulation of VDR, we wanted to determine whether dietary Vitamin D₃ protected against UVB induced tumor formation in SKH-1 mice, a model for squamous cell carcinoma development. We examined whether there was a correlation between dietary Vitamin D₃ and ΔNp63α, VDR or PTEN expression in vivo in SKH-1 mice chronically exposed to UVB radiation and fed chow containing increasing concentrations of dietary Vitamin D₃. Although we observed differential effects of the Vitamin D₃ diet on ΔNp63α and VDR expression in chronically irradiated normal mouse skin as well as UVB induced tumors, Vitamin D₃ had little effect on PTEN expression in vivo. While low-grade papillomas in mice exposed to UV and fed normal chow displayed increased levels of ΔNp63α, expression of both ΔNp63α and VDR was reduced in invasive tumors. Interestingly, in mice fed high Vitamin D₃ chow, elevated levels of ΔNp63α were observed in both local and invasive tumors but not in normal skin suggesting that oral supplementation with Vitamin D₃ may increase the proliferative potential of skin tumors by increasing ΔNp63α levels.