TRPV6 Gene Expression Research Articles

PSUN112 Expression of Vitamin D Receptor Pathway Genes Across Tissues of Individuals with Obesity

Abstract Introduction Vitamin D deficiency is a substantial comorbidity in 50% of pediatric patients and is linked with poorer health outcomes in children. Vitamin D levels are inversely related to BMI. Therefore, the prevalence of children with low vitamin D levels is increasing as the prevalence of pediatric obesity increases. Pediatric patients with obesity and vitamin D deficiency also have a uniquely increased risk of metabolic syndrome, as compared to their lean peers. However, measured levels of vitamin D correlate with other physiological markers of vitamin D effects in lean individuals but not obese individuals. Since vitamin D levels measured as 25-hydroxyvitmain D (serum 25(OH)D) reflect a storage form of vitamin D, their levels may not be a true reflection of vitamin D action in the body in an obese population. The aim of this study was to provide foundational knowledge to understand if expression of vitamin D receptor (VDR)-target genes in different metabolic pathways correlate with each other in diverse tissues, and thus may be used as a reference standard for vitamin D action in the body. Methods We performed a secondary analysis of samples obtained from 89 obese adolescents aged 12 to 18 years old that were consented under a past IRB-approved protocol. The samples were collected at the time of bariatric surgery between 2004 and 2019. Subject data included age, gender, race/ethnicity, and BMI. Samples collected included blood, intestinal, and subcutaneous adipose tissue. The tissues were analyzed via Real Time-PCR to obtain quantitative levels of VDR-target gene expression, which included TLR4, THBD, and VDR in subcutaneous adipose tissue; and TRPV6, S100G, and VDR in intestinal tissue. Gene expression levels were normalized to the average of two housekeeping genes, GAPDH and RPLPO. Blood samples were analyzed for vitamin D levels. Results VDR-target gene expression of THBD, VDR, and TLR4 in subcutaneous adipose tissue was significantly correlated (p <0.05). In intestinal tissue, we also saw significant correlation between TRPV6, S100G, and VDR (p <0.05). THBD, TLR4, TRPV6, S100G, and VDR gene expression levels from the respective tissues did not correlate with circulating serum 25(OH)D levels (p >0.05). Conclusion These important preliminary findings show VDR-target genes have correlated expression patterns within different tissues – one which stores vitamin D (adipose) and one which doesn't (intestinal). Interestingly, the VDR-target gene expression levels correlated with each other despite the genes being involved in different metabolic pathways in diverse tissues. These findings also show that VDR-target gene expression does not correlate with circulating serum 25(OH)D levels. This discrepancy supports that 25(OH)D levels do not indicate levels of vitamin D action and may not be an appropriate indicator of vitamin D deficiency in the obese population. Thus, VDR-target gene expression levels may provide a better reference standard for vitamin D action in the body. This study may provide the first step in determining a new and more accurate biomarker for vitamin D deficiency and treatment in obesity. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Methionine supplementing effects on intestine, liver and uterus morphology, and on positivity and expression of Calbindin-D28k and TRPV6 epithelial calcium carriers in laying quail in thermoneutral conditions and under thermal stress.

This study aimed to provide the performance, localization and expression of the epithelial calcium transporter channels Calbindin-D28k (Calb) and TRPV6, and of the morphology of the digestive and reproductive system of laying quail under heat stress (HS), and with methionine supplementation (MS). This study characterized the positivity (immunohistochemistry) and expression (real-time PCR) of calcium channels in the kidneys, intestine and uterus of 504 laying quails under different MS (100, 110 and 120%) and temperatures (20, 24, 28 and 32°C). The animals under HS (32°C) had lower villus height, villus:crypt ratio, and goblet cell index in the duodenum and jejunum, fewer secondary and tertiary uterine folds, smaller hepatic steatosis, and increased number of distal convoluted renal tubules (CT) positive to Calb, and increased positivity in proximal CTs. Deleterious effects of HS were minimized with MS for: duodenal crypts, number of goblet cells of the jejunum, number of uterine folds, decreased Calb positivity in intestines and kidney, increased positivity of Calb in the uterus and increased TRPV6 gene expression in the kidney (P≤0.05). Epithelial calcium transporters were altered due to less need for calcium absorption and reabsorption due to more calcium available with the MS, increasing egg production in HS and quality in termoneutrality (P≤0.05). MS further increased intestinal villus absorption area and height, increased steatosis, decreased Calb positivity in the intestine and kidney, increased uterine positivity of Calb, and increase Calb and TRPV6 expression in the kidney (P≤0.001) under thermoneutrality. It was concluded that the use of MS (120%) is justifiable in order to partially reverse the deleterious effects of HS on the production, in the epithelial calcium carriers, and in the digestory and reproductive morphology of laying quail.

Targeting 1,25(OH)2D-mediated calcium absorption machinery in proximal colon with calcitriol glycosides and glucuronides

High intestinal calcium (Ca) absorption efficiency is associated with high peak bone mass in adolescents and reduced bone loss in adulthood. Transepithelial intestinal Ca absorption is mediated by 1,25-dihydroxyvitamin D (1,25(OH)2D, calcitriol) through the vitamin D receptor (VDR). Most research on Ca absorption focuses on the proximal small intestine but evidence shows that large intestine plays a crucial role in whole body Ca homeostasis. We directly assessed and compared Ca absorption capacity at the proximal colon and duodenum using in situ ligated loops (2 mM Ca, 10 min). In C57BL/6 J mice, the proximal colon (26.2 ± 3.7 %) had comparable ability to absorb Ca as the duodenum (30.0 ± 6.7 %). In VDR knockout (KO) mice, Ca absorption efficiency was reduced by 67 % in duodenum and 48 % in proximal colon. These data suggest that large intestine could be targeted to improve Ca absorption and protect bone in at risk-groups (e.g. bariatric patients). Glycoside forms of calcitriol found in Solanum Glaucophyllum (Sg) leaf are biologically inert but can be activated in the colon upon bacterial cleavage of the glycosides. We conducted a study to test whether Sg leaf, as well as a novel, synthetic 1,3-diglucuronide form of calcitriol (1,3-diG) could target the proximal colon and upregulate genes involved in Ca absorption (i.e. Trpv6, S100g). 13-week-old female C57BL6/J mice were fed AIN93 G diet containing increasing levels of one of the two compounds for 2 weeks (delivering 0, 0.25, 0.5, 1, or 2 ng calcitriol equivalent per day). Both compounds induced a dose-dependent upregulation of Cyp24a1 and Trpv6 gene expression in the proximal colon. 1,3-diG also induced S100g gene expression in the proximal colon. Duodenal expression of Trpv6 was upregulated at higher doses of 1,3-diG but not Sg leaf. These data suggest that both glycosylated and glucuronidated calcitriol could be used to target the proximal colon but that dosing must be optimized to limit systemic effects that could cause hypercalcemia. Future studies will test the translational potential of these compounds to determine if they can increase Ca absorption at proximal colon and whether this can help protect bone.

53 Pre-implantation exposure to bisphenol A and 4-tert-octylphenol result in disruption of calcium channels

Miscarriage due to blastocyst implantation failure occurs in up to two-thirds of all miscarriage cases in humans. Calcium (Ca2+) has been shown to involve many cellular signal transduction pathways as well as regulation of cell adhesion, which is necessary for the physiology process of endometrial epithelial cell transformation and stromal cell decidualization during embryo implantation. Exposure to endocrine-disrupting chemicals (ED) can regulate the expression of genes associated with calcium transport in during pregnancy such as TRPV5, TRPV6, PMCA, and NCX1. Additionally, exposure to ED during early gestation results in disrupted intrauterine implantation, uterine receptive, leading to implantation failure. In this study, oestrogen (E2), bisphenol A (BPA), octylphenol (OP), and/or ICI 182,780 (oestrogen receptor antagonist, ICI) were injected subcutaneously from gestation Day 1 to gestation Day 3 post coitus. The number of implantation sites was significantly lower in the OP group, and no implantation site was observed in the E2 and ED+ICI groups. There were differences in the expression of calcium transient transport channel between maternal uterine and implantation. The level of TRPV6 and TRPV5 mRNA and protein was significantly increased by ED and/or ICI treatment in the uterus. The levels of TRPV5 and TRPV6 gene expression were significantly increased by ED with/without ICI treatment in the uterus. However, TRPV5 and TRPV6 gene expression was significantly lower in implantation site samples. The NCX1 and PMCA1 mRNA levels were significantly decreased by OP and BPA in the implantation site samples. Both mRNA and protein levels of MUC1 were markedly higher in all groups, except the BPA group when compared with the vehicle group in the uterus. The LIF and HOXA-10 mRNA were significantly low in E2; BPA+ICI; OP; and/or ICI in both the uterus and implantation site. Expression of the oestrogen receptor (ERa) and progesterone receptor (PR) was significantly lower in all groups except the BPA group when compared with the vehicle group. Taken together, E2, BPA, and OP disrupt the success of implantation through altered expression of calcium transport genes.

Effects of Bisphenol A and 4-tert-Octylphenol on Embryo Implantation Failure in Mouse.

Miscarriage due to blastocyst implantation failure occurs in up to two-thirds of all human miscarriage cases. Calcium ion has been shown to be involved in many cellular signal transduction pathways as well as in the regulation of cell adhesion, which is necessary for the embryo implantation process. Exposure to endocrine-disrupting chemicals (EDs) during early gestation results in disruption of intrauterine implantation and uterine reception, leading to implantation failure. In this study, ovarian estrogen (E2), bisphenol A (BPA), or 4-tert-octylphenol (OP), with or without ICI 182,780 (ICI) were injected subcutaneously from gestation day 1 to gestation day 3 post-coitus. The expression levels of the calcium transport genes were assessed in maternal uteri and implantation sites. The number of implantation sites was significantly low in the OP group, and implantation sites were absent in the E2, ICI and EDs + ICI groups. There were different calcium transient transport channel expression levels in uterus and implantation site samples. The levels of TRPV5 and TRPV6 gene expression were significantly increased by EDs with/without ICI treatment in utero. Meanwhile, TRPV5 and TRPV6 gene expression were significantly lower in implantation sites samples. NCX1 and PMCA1 mRNA levels were significantly decreased by OP and BPA in the implantation site samples. Compared to vehicle treatment in the uterus, both the MUC1 mRNA and protein levels were markedly high in all but the BPA group. Taken together, these results suggest that both BPA and OP can impair embryo implantation through alteration of calcium transport gene expressions and by affecting uterine receptivity.

Relationship Between Yolk Sac Liquid Crystal Formation and Calcium Transport in Pigeon Egg Yolk Sac Endoderm

During the incubation of pigeon eggs, the yolk is transformed from a disordered to an ordered state before being absorbed, and the liquid crystals formed the ordered structure via self-assembly in the yolk. In this study, the relationship between the enzymes (ACPase and Ca-ATPase) and liquid crystal formation within the pigeon endoderm was determined. The results showed that ACPase is critical for transforming the yolk into liquid crystals, which originate mainly from the endoderm. The formation of lamellar structures in the yolk was closely associated with the hydrolytic activity of the enzymes. Additionally, a large amount of Ca-ATPase was expressed in the lamellar structures, indicating modulatory effects of Ca-ATPase on the precipitation of calcium carbonate and appearance of lamellar spherical structures. The liquid crystals containing transport enzymes, which formed as a result of laminarization, serve as reaction sites as well as a reactive source of calcium. The transient receptor potential cation channel subfamily V, member 6 (TRPV6) is an epithelial calcium channel. The present study aimed to elucidate the relationship between yolk sac liquid crystals and calcium transport by investigating the regulatory mechanisms underlying TRPV6 gene expression in the pigeon endoderm. The pattern of expression of TRPV6 observed in this study suggests that yolk sac liquid crystals participate in calcium transport in the endoderm.

Abstract LB-128: High prevalence of elevated TRPV6 mRNA in pancreatic ductal adenocarcinoma

Abstract SOR-C13, a 13-mer peptide derived from soricidin, the paralytic protein component of saliva of the Northern Short-tailed shrew, just completed an open-label, all comers phase I clinical trial for the treatment of epithelial-derived cancers. SOR-C13 is a first-in-class drug candidate that specifically targets and inhibits the TRPV6 calcium channel - a recognized oncochannel over-expressed in a number of epithelial cancers (e.g. breast, ovarian, prostate). TRPV6 over-expression is associated with a poor prognosis particularly with breast and prostate cancers. SOR-C13 is the first TRPV6-targeting drug to enter clinical development. Pancreatic patients were enrolled in the phase I clinical trial for SOR-C13 and for this reason the TRPV6 gene expression in pancreatic tumour biopsies was assessed. Association of TRPV6 oncochannel expression with aggressive pancreatic adenocarcinoma compared to neuroendocrine tumours has never been investigated. Pancreatic adenocarcinoma represents 90% of the pancreatic tumours and is associated with a poor prognosis compared to neuroendocrine tumours. TRPV6 gene expression was assessed in 19 pancreatic tumor biopsies including five adenocarcinoma (Grade I to III), one acinar cell (exocrine), and 13 neuroendocrine (Islet cell) tumors by two-step RT-qPCR. TRPV6 and β-actin (reference gene) mRNA expression was evaluated in duplicate in a reproducible TRPV6 plus β-actin duplex two-step RT-qPCR assay. The acinar cell, and 69% (9/13) of the neuroendocrine tumors had no detectable TRPV6 mRNA expression with >90% of neuroendocrine tumors (12/13) having very little TRPV6 mRNA expression (<30 copies/ng RNA). Of the five pancreatic adenocarcinoma biopsies 4/5 (80%) had 67-285 copies of TRPV6 mRNA/ng RNA, indicating a high level of TRPV6 expression in this type of aggressive pancreatic cancer. The β-actin CT was between 24 and 30 for all tumours (mean 28.5 ± 1.2) indicating that the lack TRPV6 amplification in a majority of the neuroendocrine tumors was not due to RT-qPCR failure or a lack of quality of the biopsy cDNA. Results indicate that drugs specifically targeting TRPV6 oncochannel, like SOR-C13, have the potential to be used to treat the high medical need in pancreatic ductal adenocarcinoma. Citation Format: Tyler Lutes, Dominique Dugourd, Michelle Davey, Christopher Rice, Stephanie St-Pierre, Jack M. Stewart. High prevalence of elevated TRPV6 mRNA in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-128.

TRPV6 channels.

TRPV6 (former synonyms ECAC2, CaT1, CaT-like) displays several specific features which makes it unique among the members of the mammalian Trp gene family (1) TRPV6 (and its closest relative, TRPV5) are the only highly Ca(2+)-selective channels of the entire TRP superfamily (Peng et al. 1999; Wissenbach et al. 2001; Voets et al. 2004). (2) Translation of Trpv6 initiates at a non-AUG codon, at ACG, located upstream of the annotated AUG, which is not used for initiation (Fecher-Trost et al. 2013). The ACG codon is nevertheless decoded by methionine. Not only a very rare event in eukaryotic biology, the full-length TRPV6 protein existing in vivo comprises an amino terminus extended by 40 amino acid residues compared to the annotated truncated TRPV6 protein which has been used in most studies on TRPV6 channel activity so far. (In the following numbering occurs according to this full-length protein, with the numbers of the so far annotated truncated protein in brackets). (3) Only in humans a coupled polymorphism of Trpv6 exists causing three amino acid exchanges and resulting in an ancestral Trpv6 haplotype and a so-called derived Trpv6 haplotype (Wissenbach et al. 2001). The ancestral allele encodes the amino acid residues C197(157), M418(378) and M721(681) and the derived alleles R197(157), V418(378) and T721(681). The ancestral haplotype is found in all species, the derived Trpv6 haplotype has only been identified in humans, and its frequency increases with the distance to the African continent. Apparently the Trpv6 gene has been a strong target for selection in humans, and its derived variant is one of the few examples showing consistently differences to the orthologues genes of other primates (Akey et al. 2004, 2006; Stajich and Hahn 2005; Hughes et al. 2008). (4) The Trpv6 gene expression is significantly upregulated in several human malignancies including the most common cancers, prostate and breast cancer (Wissenbach et al. 2001; Zhuang et al. 2002; Fixemer et al. 2003; Bolanz et al. 2008). (5) Male mice lacking functional TRPV6 channels are hypo-/infertile making TRPV6 one of the very few channels essential for male fertility (Weissgerber et al. 2011, 2012).

Sulforaphane and trichostatin A histone deacetylase inhibitors increase vitamin D‐induced CYP24 expression in intestinal cells

Epigenetic changes, such as acetylation of nuclear histone proteins, can be induced by bioactive food components and alter chromatin structure and the expression of certain genes. Sulforaphane (SFN) and trichostatin A (TSA) have histone deacetylase inhibitor (HDACI) activity. We hypothesized that HDACI treatment would increase vitamin D‐induced CYP24 and TRPV6 gene expression. We tested the effect of four dosages of SFN (1, 2.5, 5 and 10 μmol/L) and one dose of TSA (1 μmol/L) with and without the presence of 10 nmol/L 1, 25‐dihydroxyvitamin D (1, 25 (OH)2D) for 24h on the expression of CYP24 and TRPV6 in the Caco‐2 human intestinal cell model. Western blot analysis showed an increase in the protein expression levels of CYP24 with SFN and 1,25 (OH)2D. Realtime qPCR was used to calculate the relative change in mRNA expression of CYP24 and TRPV6. TSA alone increased (p<.05) CYP24 mRNA expression. 1,25 (OH)2D increased CYP24 and TRPV6 mRNA expression in Caco‐2 cells. TSA also increased vitamin D‐induced CYP24 mRNA expression, but vitamin D‐induced TRPV6 expression was not affected by HDACI treatment. Our results demonstrate that HDACIs can differentially affect the expression of certain vitamin D‐dependent genes in Caco‐2 intestinal cells. Additional studies are needed to determine HDACI effects on vitamin D‐related functions in vivo. This work was supported by a Massachusetts Experiment Station HATCH grant MAS000902

Alterations in the viability, cell cycle and TRPV6 gene expression induced by calcitriol and BSO in breast cancer cells

Alterations in the viability, cell cycle and TRPV6 gene expression induced by calcitriol and BSO in breast cancer cells

Intestinal Vitamin D Receptor Is Required for Normal Calcium and Bone Metabolism in Mice

Vitamin D receptor (VDR)-knockout mice develop severe hypocalcemia and rickets, accompanied by disruption of active intestinal calcium absorption. To specifically study the effects of VDR in intestinal calcium absorption, we investigated whether restoration of intestinal VDR is sufficient to recover the phenotype of VDR-knockout mice. We generated mice with intestine-specific transgenic expression of human VDR and crossed them to VDR knockout mice. The intestine, kidney, and bone phenotypes of the VDR- knockout mice with intestine-specific expression of human VDR (knockout/transgenic [KO/TG]) were analyzed. Transgenic expression of VDR in the intestine of VDR-knockout mice normalized duodenal vitamin D-regulated calcium absorption as well as vitamin D-regulated calcium binding protein D9k and TRPV6 gene expression in the duodenum and proximal colon. As a result, animal growth and the serum levels of calcium and parathyroid hormone were normalized in KO/TG mice. Other phenotypes were revealed when calcium metabolism was normalized in KO/TG mice: serum 1,25 dihydroxyvitamin D levels were higher in KO/TG mice than normal mice owing to reduced renal expression of the vitamin D-degrading enzyme CYP24, urinary calcium excretion was higher and associated with lower renal calcium binding protein D9k and calcium binding protein D28k than normal mice, and bone density and volume increased in KO/TG compared with normal mice owing to increased mineral apposition rate and osteoblast number. Intestinal VDR and vitamin D-regulated intestinal calcium absorption are critical for controlling whole-body calcium metabolism in growing mice. Normalizing intestinal calcium absorption and metabolism reveals essential roles for VDR in control of bone formation and renal control of serum 1,25(OH)2D and urinary calcium excretion.