TRPV6 Research Articles

Abstract 1211: Ecological model-generated hypothesis for high prostate cancer incidence in African-Americans: TRPV6a gene variant and calcium-ion hypersensitivity

Abstract INTRODUCTION: This Ecological Model System (EMS) applies an evidence-based hypothesis to a health disparities conundrum posed by low-calcium-consuming African-American males in the sizable medical literature that attributes metastatic Prostate Cancer (PCa) for which this group is at high risk, to calcium over-consumption. This EMS identifies the ethnic-specific TRPV6 calcium ion channel,because of its unusually high calcium absorption capacity relative to the non-African variant, as the culprit. While it allowed the Niger-Kordofanian West African ancestors of black Americans to maintain strong bones on a 200 mg/ca/day diet, it is oncologically maladaptive for U.S. blacks. In America's high calcium, dairy food culture, the TRPV6a variant allows blacks, who are generally lactase non-persistent, to nevertheless absorb more of the mineral than whites and excrete less in their urine. These excess free calcium ions appear to initiate cell proliferation, leading to metastatic PCa, in which the TRPV6 gene becomes over-expressed. METHODS: To generate an algorithm-driven informative matrix, Medline and JSTOR searches were mined using keywords such as: metastatic prostatic cancer, androgen-castration-resistant, Niger-Kordofanian West Africans, TRPV6, TRPV5-renal excretion, TRPV6 over-expression biomarker. Secondary data sets from (1) “Prostate, Lung, Colorectal, Ovarian Cancer Screening Trial,” conducted between 1993 and 2001, 38,349 males (ages 55-74 years); (2) “California Collaborative Prostate Cancer Study”, a study of 4321 African-Americans and non-Hispanic Whites was analyzed using multivariable conditional logistic regression. RESULTS: This EMS reveals that at 400 mg/day/ca, metastatic PCa is observed in 17% of Blacks, but 0% of American Whites. 100% of the Black incidence is below 1150 mg/calcium intake, while 59% of White incidence from 1250-2000 mg. SUMMARY: Because the African TRPV6a calcium ion channel variant is more absorbent of free calcium ions than its non-African counterpart, this Ecological Model shows that carriers of this variant are at increased risk of metastatic PCa when exposed to high calcium food environments. CONCLUSION: Black hypersensitivity to calcium triggers metastatic PCa caused by high-calcium-absorbing African TRPV6a calcium ion channel variant. This EMS further hypothesizes that the high susceptibility of Blacks to other TRPV6-expressing cancers (e.g.triple negative breast cancer and colorectal cancer) share the same pathogenesis. This Model can be applied as well to generating hypotheses for STEM basic and translational research regarding Type II diabetes, hypertension, kidney disease. Citation Format: Constance B. Hilliard. Ecological model-generated hypothesis for high prostate cancer incidence in African-Americans: TRPV6a gene variant and calcium-ion hypersensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1211.

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.

Leukocyte TRP channel gene expressions in\xa0patients with non-valvular atrial fibrillation

Atrial fibrillation (AF) is the most common arrhythmia in clinical practice and is a major cause of morbidity and mortality. The upregulation of TRP channels is believed to mediate the progression of electrical remodelling and the arrhythmogenesis of the diseased heart. However, there is limited data about the contribution of the TRP channels to development of AF. The aim of this study was to investigate leukocyte TRP channels gene expressions in non-valvular atrial fibrillation (NVAF) patients. The study included 47 NVAF patients and 47 sex and age matched controls. mRNA was extracted from blood samples, and real-time polymerase chain reaction was performed for gene expressions by using a dynamic array system. Low levels of TRP channel expressions in the controls were markedly potentiated in NVAF group. We observed marked increases in MCOLN1 (TRPML1), MCOLN2 (TRPML2), MCOLN3 (TRPML3), TRPA1, TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7, TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, TRPV6, and PKD2 (TRPP2) gene expressions in NVAF patients (P < 0.05). However, there was no change in PKD1 (TRPP1) gene expression. This is the first study to provide evidence that elevated gene expressions of TRP channels are associated with the pathogenesis of NVAF.

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 &amp;gt;90% of neuroendocrine tumors (12/13) having very little TRPV6 mRNA expression (&amp;lt;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.

P3-4-4 - Investigation of the Relationship Between Breast Cancer and Transient Receptor Potential (Trp) Genes

Background: Breast cancer is the most common cancer type among woman. Ion channels are important for different physiologic functions as muscle contraction and hormone secretion. Recently the effect of Transient Receptor Potential (TRP) channels on calcium balance, cell proliferation, cell differantiation, tumor invasion, oncogen regulation and resistance to apoptotic cell death has been demonstrated. Some members of TRP family as TRPV6, TRPM1, TRPM8 and TRPM5 has been directly associated with cancer. The aim of this study is to determine the importance of TRP channels in breast cancer.Methods: Thirty two breast cancer patients were enrolled. Normal breast tissue and breast cancer tissue has been taken from each patient during operation. Tissues has been protected in liquid nitrogen. RNA isolation from tissues was made with RNA isolation kit and cDNA has been derived with reverse transcriptase reaction (PCR) from RNA. Expression of 20 genes containing TRPC, TRPM, TRPV and TRPA genes has been investigated with Biomark Fludigm nano Real Time PCR tool.Results: The expression of TRPM1, TRPC7 and TRPC5 genes has not been observed in normal breast and breast cancer tissues. Compared to normal breast tissue TRPMA1, TRMPV5, TRPM5 and TRPV6 genes expression was at high levels in breast cancer tissues. There was no expression of TRPMA1, TRMPV5, TRPM5 and TRPV6 genes in normal breast tissues.Conclusion: In this study the importance of TRP channels in breast cancer has been evaluated. Expression of genes for regulating the TRP channel proteins could be an important candidate for breast cancer diagnosis and treatment. Especially expression of TRPMA1, TRMPV5, TRPM5 and TRPV6 genes has been determined at high levels in breast cancer tissues. Evaluation of the clinical effects of these genes on breast cancer progression can be used at new researchs for diagnosis and treatment options of breast cancer.

The transient receptor potential cation channel subfamily V member 6 (TRPV6): genetics, biochemical properties, and functions of exceptional calcium channel proteins.

The transient receptor potential cation channel subfamily V member 6 (TRPV6) gene and cDNA were identified 15 years ago and exceptional observations on TrpV6 proteins and their function as a Ca(2+)-selective cation channel have been made since then. In this review we will summarize recent studies regarding the genetics, biochemical properties, and physiological functions of murine and human TrpV6 channel proteins. We will focus on TRPV6 gene polymorphisms, the start of TRPV6 translation at a non-AUG codon and the functions of TRPV6 in intestinal Ca(2+) uptake, sperm maturation, and male fertility.

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).

TRPV5: a Ca(2+) channel for the fine-tuning of Ca(2+) reabsorption.

TRPV5 is one of the two channels in the TRPV family that exhibit high selectivity to Ca(2+) ions. TRPV5 mediates Ca(2+) influx into cells as the first step to transport Ca(2+) across epithelia. The specialized distribution in the distal tubule of the kidney positions TRPV5 as a key player in Ca(2+) reabsorption. The responsiveness in expression and/or activity of TRPV5 to hormones such as 1,25-dihydroxyvitamin D3, parathyroid hormone, estrogen, and testosterone makes TRPV5 suitable for its role in the fine-tuning of Ca(2+) reabsorption. This role is further optimized by the modulation of TRPV5 trafficking and activity via its binding partners; co-expressed proteins; tubular factors such as calbindin-D28k, calmodulin, klotho, uromodulin, and plasmin; extracellular and intracellular factors such as proton, Mg(2+), Ca(2+), and phosphatidylinositol-4,5-bisphosphate; and fluid flow. These regulations allow TRPV5 to adjust its overall activity in response to the body's demand for Ca(2+) and to prevent kidney stone formation. A point mutation in mouse Trpv5 gene leads to hypercalciuria similar to Trpv5 knockout mice, suggesting a possible role of TRPV5 in hypercalciuric disorders in humans. In addition, the single nucleotide polymorphisms in Trpv5 gene prevalently present in African descents may contribute to the efficient renal Ca(2+) reabsorption among African descendants. TRPV5 represents a potential therapeutic target for disorders with altered Ca(2+) homeostasis.

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&lt;.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

Autosomal Dominant Hypercalciuria in a Mouse Model Due to a Mutation of the Epithelial Calcium Channel, TRPV5

Hypercalciuria is a major cause of nephrolithiasis, and is a common and complex disorder involving genetic and environmental factors. Identification of genetic factors for monogenic forms of hypercalciuria is hampered by the limited availability of large families, and to facilitate such studies, we screened for hypercalciuria in mice from an N-ethyl-N-nitrosourea mutagenesis programme. We identified a mouse with autosomal dominant hypercalciuria (HCALC1). Linkage studies mapped the Hcalc1 locus to a 11.94 Mb region on chromosome 6 containing the transient receptor potential cation channel, subfamily V, members 5 (Trpv5) and 6 (Trpv6) genes. DNA sequence analysis of coding regions, intron-exon boundaries and promoters of Trpv5 and Trpv6 identified a novel T to C transition in codon 682 of TRPV5, mutating a conserved serine to a proline (S682P). Compared to wild-type littermates, heterozygous (Trpv5 682P/+) and homozygous (Trpv5 682P/682P) mutant mice had hypercalciuria, polyuria, hyperphosphaturia and a more acidic urine, and ∼10% of males developed tubulointerstitial nephritis. Trpv5 682P/682P mice also had normal plasma parathyroid hormone but increased 1,25-dihydroxyvitamin D3 concentrations without increased bone resorption, consistent with a renal defect for the hypercalciuria. Expression of the S682P mutation in human embryonic kidney cells revealed that TRPV5-S682P-expressing cells had a lower baseline intracellular calcium concentration than wild-type TRPV5-expressing cells, suggesting an altered calcium permeability. Immunohistological studies revealed a selective decrease in TRPV5-expression from the renal distal convoluted tubules of Trpv5 682P/+ and Trpv5 682P/682P mice consistent with a trafficking defect. In addition, Trpv5682P/682P mice had a reduction in renal expression of the intracellular calcium-binding protein, calbindin-D28K, consistent with a specific defect in TRPV5-mediated renal calcium reabsorption. Thus, our findings indicate that the TRPV5 S682P mutant is functionally significant and study of HCALC1, a novel model for autosomal dominant hypercalciuria, may help further our understanding of renal calcium reabsorption and hypercalciuria.

Calcium Channel TRPV6 as a Potential Therapeutic Target in Estrogen Receptor–Negative Breast Cancer

Calcium signaling is a critical regulator of cell proliferation. Elevated expression of calcium channels and pumps is a characteristic of some cancers, including breast cancer. We show that the plasma membrane calcium channel TRPV6, which is highly selective for Ca(2+), is overexpressed in some breast cancer cell lines. Silencing of TRPV6 expression in a breast cancer cell line with increased endogenous TRPV6 expression leads to a reduction in basal calcium influx and cellular proliferation associated with a reduction in DNA synthesis. TRPV6 gene amplification was identified as one mechanism of TRPV6 overexpression in a subset of breast cancer cell lines and breast tumor samples. Analysis of two independent microarray expression datasets from breast tumor samples showed that increased TRPV6 expression is a feature of estrogen receptor (ER)-negative breast tumors encompassing the basal-like molecular subtype, as well as HER2-positive tumors. Breast cancer patients with high TRPV6 levels had decreased survival compared with patients with low or intermediate TRPV6 expression. Our findings suggest that inhibitors of TRPV6 may offer a novel therapeutic strategy for the treatment of ER-negative breast cancers.

Villin promoter-mediated transgenic expression of transient receptor potential cation channel, subfamily V, member 6 (TRPV6) increases intestinal calcium absorption in wild-type and vitamin D receptor knockout mice.

Transient receptor potential cation channel, subfamily V, member 6 (TRPV6) is an apical membrane calcium (Ca) channel in the small intestine proposed to be essential for vitamin D-regulated intestinal Ca absorption. Recent studies have challenged the proposed role for TRPV6 in Ca absorption. We directly tested intestinal TRPV6 function in Ca and bone metabolism in wild-type (WT) and vitamin D receptor knockout (VDRKO) mice. TRPV6 transgenic mice (TG) were made with intestinal epithelium-specific expression of a 3X Flag-tagged human TRPV6 protein. TG and VDRKO mice were crossed to make TG-VDRKO mice. Ca and bone metabolism was examined in WT, TG, VDRKO, and TG-VDRKO mice. TG mice developed hypercalcemia and soft tissue calcification on a chow diet. In TG mice fed a 0.25% Ca diet, Ca absorption was more than three-fold higher and femur bone mineral density (BMD) was 26% higher than WT. Renal 1α hydroxylase (CYP27B1) mRNA and intestinal expression of the natural mouse TRPV6 gene were reduced to <10% of WT but small intestine calbindin-D(9k) expression was elevated >15 times in TG mice. TG-VDRKO mice had high Ca absorption that prevented the low serum Ca, high renal CYP27B1 mRNA, low BMD, and abnormal bone microarchitecture seen in VDRKO mice. In addition, small intestinal calbindin D(9K) mRNA and protein levels were elevated in TG-VDRKO. Transgenic TRPV6 expression in intestine is sufficient to increase Ca absorption and bone density, even in VDRKO mice. VDR-independent upregulation of intestinal calbindin D(9k) in TG-VDRKO suggests this protein may buffer intracellular Ca during Ca absorption. © 2012 American Society for Bone and Mineral Research.

Excision of Trpv6 Gene Leads to Severe Defects in Epididymal Ca2+ Absorption and Male Fertility Much Like Single D541A Pore Mutation

Replacement of aspartate residue 541 by alanine (D541A) in the pore of TRPV6 channels in mice disrupts Ca(2+) absorption by the epididymal epithelium, resulting in abnormally high Ca(2+) concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilization capacity, raising the possibility of residual activity of channels formed by TRPV6(D541A) proteins (Weissgerber, P., Kriebs, U., Tsvilovskyy, V., Olausson, J., Kretz, O., Stoerger, C., Vennekens, R., Wissenbach, U., Middendorff, R., Flockerzi, V., and Freichel, M. (2011) Sci. Signal. 4, ra27). It is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared with the deletion of the corresponding protein. Therefore, we generated TRPV6-deficient mice (Trpv6(-/-)) by deleting exons encoding transmembrane domains with the pore-forming region and the complete cytosolic C terminus harboring binding sites for TRPV6-associated proteins that regulate its activity and plasma membrane anchoring. Using this strategy, we aimed to determine whether the TRPV6(D541A) pore mutant still contributes to residual channel activity and/or channel-independent functions in vivo. Trpv6(-/-) males reveal severe defects in fertility and motility and viability of sperm and a significant increase in epididymal luminal Ca(2+) concentration that is mirrored by a lack of Ca(2+) uptake by the epididymal epithelium. Therewith, Trpv6 excision affects epididymal Ca(2+) handling and male fertility to the same extent as the introduction of the D541A pore mutation, arguing against residual functions of the TRPV6(D541A) pore mutant in epididymal epithelial cells.

148 DIFFERENTIAL REGULATION OF CALCIUM TRANSPORT GENES, I.E., Na+/Ca2+ EXCHANGERS, TRANSIENT RECEPTOR POTENTIAL CATION CHANNEL 6 AND CALBINDINS, IN THE DIVERSE PLACENTAL TISSUES OF CALBINDIN-D9k AND -28k KNOCKOUT MICE VIA PUTATIVE STEROIDS

During pregnancy, the placenta represents the establishment of an intimate connection between mother and fetus that is specific to mammals. Calbindins [Calbindin-D9k (CaBP-9k) and -D28k (CaBP-28k)] are proteins possessing EF-hand motifs that have a high affinity for Ca2+ ions and play an important role in the regulation and buffering of Ca2+ in the various tissues. Many types of calcium channels, intracellular calcium binding proteins, Na+/Ca2+ exchangers (NCX) and transient receptor potential cation channels (TRPV) have been found in the placenta. In this study, the calcium channel in maternal-fetal Ca2+ transport was investigated using the phenotypes of wild-type, CaBP-9k, CaBP-28k and CaBP-9k/28k knockout (KO) mouse models. Expressions of calcium transport genes in 3 dissected sections of placenta (MP: maternal, CP: central, FP: fetal) were examined by real-time RT-PCR (RT-qPCR) and Western blot analysis at gestational Day 19 in these mice. The level of TRPV6 mRNA and protein was highest in the MP and CP of CaBP-28k KO mice and the FP of CaBP-9k KO mice compared with other sections of KO mice. The level of CaBP-9k was significantly induced in CaBP-28k KO mice in MP, CP and FP compared with in WT mice, which levels were elevated from maternal to fetal sections. The expression of CaBP-28k mRNA and protein was reduced in CaBP-9k KO mice compared with WT in the 3 sections of placenta. The expression of NCX1 mRNA and protein was higher in all KO mice than in WT in MP and NCX1 was highest in CaBP-28k KO mice in CP, but strong in CaBP-9k KO mice in FP compared with other strains. These results indicate that TRPV6 and NCX1 participate in transferring calcium ions between maternal and fetal compartments and alteration of CaBP-9k/28k is involved in the intracellular Ca2+ buffering system among WT and KO mice. These results taken together indicate that TRPV6 and CaBP-9k genes may play a role as a key element in controlling placental calcium transport during pregnancy.

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

Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2

Nedd4-2 is an archetypal HECT ubiquitin E3 ligase that disposes target proteins for degradation. Because of the proven roles of Nedd4-2 in degradation of membrane proteins, such as epithelial Na(+) channel, we examined the effect of Nedd4-2 on the apical Ca(2+) channel TRPV6, which is involved in transcellular Ca(2+) transport in the intestine using the Xenopus laevis oocyte system. We demonstrated that a significant amount of Nedd4-2 protein was distributed to the absorptive epithelial cells in ileum, cecum, and colon along with TRPV6. When co-expressed in oocytes, Nedd4-2 and, to a lesser extent, Nedd4 down-regulated the protein abundance and Ca(2+) influx of TRPV6 and TRPV5, respectively. TRPV6 ubiquitination was increased, and its stability was decreased by Nedd4-2. The Nedd4-2 inhibitory effects on TRPV6 were partially blocked by proteasome inhibitor MG132 but not by the lysosome inhibitor chloroquine. The rate of TRPV6 internalization was not significantly altered by Nedd4-2. The HECT domain was essential to the inhibitory effect of Nedd4-2 on TRPV6 and to their association. The WW1 and WW2 domains interacted with TRPV6 terminal regions, and a disruption of the interactions by D204H and D376H mutations in the WW1 and WW2 domains increased TRPV6 ubiquitination and degradation. Thus, WW1 and WW2 may serve as a molecular switch to limit the ubiquitination of TRPV6 by the HECT domain. In conclusion, Nedd4-2 may regulate TRPV6 protein abundance in intestinal epithelia by controlling TRPV6 ubiquitination.

The Identification of Histidine 712 as a Critical Residue for Constitutive TRPV5 Internalization

The epithelial Ca(2+) channel TRPV5 constitutes the apical entry gate for Ca(2+) transport in renal epithelial cells. Ablation of the trpv5 gene in mice leads to a reduced Ca(2+) reabsorption. TRPV5 is tightly regulated by various calciotropic hormones, associated proteins, and other factors, which mainly affect channel activity via the C terminus. To further identify the role of the C terminus in TRPV5 regulation, we expressed channels harboring C-terminal deletions and studied channel activity by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) using fura-2 analysis. Removal of amino acid His(712) elevated the [Ca(2+)](i), indicating enlarged TRPV5 activity. In addition, substitution of the positively charged His(712) for a negative (H712D) or neutral (H712N) amino acid also stimulated TRPV5 activity. This critical role of His(712) was confirmed by patch clamp analysis, which demonstrates increased Na(+) and Ca(2+) currents for TRPV5-H712D. Cell surface biotinylation studies revealed enhanced plasma membrane expression of TRPV5-H712D as compared with wild-type (WT) TRPV5. This elevated plasma membrane presence also was observed with the Ca(2+)-impermeable TRPV5-H712D and TRPV5-WT pore mutants, demonstrating that the elevation is not due to the increased [Ca(2+)](i). Finally, using an internalization assay, we demonstrated a delayed cell surface retrieval for TRPV5-H712D, likely causing the increase in plasma membrane expression. Together, these results demonstrate that His(712) plays an essential role in plasma membrane regulation of TRPV5 via a constitutive endocytotic mechanism.

TRPV6 alleles do not influence prostate cancer progression

BackgroundThe transient receptor potential, subfamily V, member 6 (TRPV6) is a Ca2+ selective cation channel. Several studies have shown that TRPV6 transcripts are expressed in locally advanced prostatic adenocarcinoma, metastatic and androgen-insensitive prostatic lesions but are undetectable in healthy prostate tissue and benign prostatic hyperplasia. Two allelic variants of the human trpv6 gene have been identified which are transcribed into two independent mRNAs, TRPV6a and TRPV6b. We now asked, whether the trpv6a allele is correlated with the onset of prostate cancer, with the Gleason score and the tumour stage.MethodsGenomic DNA of prostate cancer patients and control individuals was isolated from resections of prostatic adenocarcinomas and salivary fluid respectively. Genotyping of SNPs of the TRPV6 gene was performed by restriction length polymorphism or by sequencing analysis. RNA used for RT-PCR was isolated from prostate tissue. Data sets were analyzed by Chi-Square test.ResultsWe first characterized in detail the five polymorphisms present in the protein coding exons of the trpv6 gene and show that these polymorphisms are coupled and are underlying the TRPV6a and the TRPV6b variants. Next we analysed the frequencies of the two TRPV6 alleles using genomic DNA from saliva samples of 169 healthy individuals. The homozygous TRPV6b genotype predominated with 86%, whereas no homozygous TRPV6a carriers could be identified. The International HapMap Project identified a similar frequency for an Utah based population whereas in an African population the a-genotype prevailed. The incidence of prostate cancer is several times higher in African populations than in non-African and we then investigated the TRPV6a/b frequencies in 141 samples of prostatic adenocarcinoma. The TRPV6b allele was found in 87% of the samples without correlation with Gleason score and tumour stage.ConclusionOur results show that the frequencies of trpv6 alleles in healthy control individuals and prostate cancer patients are not significantly different. Although expression of trpv6 transcripts correlates with aggressive potential of prostate cancer, the TRPV6 genotype does not correlate with the onset of prostate cancer, with the Gleason score and the tumour stage.

Human duodenum responses to vitamin D metabolites of TRPV6 and other genes involved in calcium absorption

Calcium absorption by the intestine is necessary for bone mineralization. Much has been learned about this process and the role of vitamin D metabolites in gene transcription from animal studies, but the molecular mechanisms in humans are less well understood. We have used samples of normal human duodenal mucosa, obtained at endoscopy, to investigate the effects of the vitamin D metabolites, 1α-dihydroxycholecalciferol [1,25(OH)2D3] and 25-hydroxycholecalciferol (25OHD), on transcripts on genes involved in calcium absorption and vitamin D metabolism. TRPV6 transcripts were significantly higher after incubation for 6 h with 1,25(OH)2D3 (10−9 mol/l) than after control incubations (median difference 3.1-fold, P < 0.001). Unexpectedly, TRPV6 expression was also higher (2.4-fold, P < 0.02) after incubation with 25OHD (10−7 mol/l). Transcripts for the calcium-ATPase, PMCA1, were significantly higher with 1,25(OH)2D3; CYP24 transcripts were reliably detected after incubation with either metabolite, but calbindin-D9k transcripts were unaffected. The response of TRPV6 to 25OHD and the expression of transcripts for CYP27B1, the 25OHD-1α-hydroxylase, were significantly correlated (r = 0.82, P < 0.02). Basal duodenal expression of TRPV6 and CYP27B1 were significantly associated (r = 0.72, P < 0.001) in a separate previously reported series of subjects. Multiple regression analysis of the associations with basal duodenal TRPV6 expression identified CYP27B1 expression and serum 1,25(OH)2D as major factors. Expression of the CYP27B1 protein was demonstrated immunohistochemically in duodenal mucosa. This study has shown that human duodenal TRPV6, PMCA1, and CYP24 transcripts respond rapidly to 1,25(OH)2D3 and provides evidence suggesting that local duodenal production of 1,25(OH)2D3 by 25OHD-1α-hydroxylase may have a role in human calcium absorption.

Uterine and placental expression of TRPV6 gene is regulated via progesterone receptor- or estrogen receptor-mediated pathways during pregnancy in rodents

Transient receptor potential cation channel, subfamily V, member 6 (TRPV6) is an epithelial Ca2+ channel protein expressed in calcium absorbing organs. In the present study, we investigated the expression and regulation of uterine and placental TRPV6 during gestation in rodents. Uterine TRPV6 peaked at pregnancy day (P) 0.5, P5.5 and, P13.5 and was detected in uterine epithelium and glands of rats, while placental TRPV6 mRNA levels increased in mid-gestation. Uterine and placental TRPV6 mRNA levels in rats appear to cyclically change during pregnancy, suggesting that TRPV6 may participate in the implantation process. In addition, uterine TRPV6 mRNA is only expressed in placenta-unattached areas of the uterus, and uterine TRPV6 immunoreactivity was observed in luminal and glandular epithelial cells. In the placenta, TRPV6 was detected in the labyrinth and spongy zone. These results may indicate that TRPV6 has at least two functions: implantation of the embryo and maintenance of pregnancy. To investigate the pathway(s) mediating TRPV6 expression in rodents, anti-steroid hormone antagonists were injected prior to maximal TRPV6 expression. In rats, TRPV6 expression was reduced by RU486 (an anti-progesterone) through progesterone receptors, and ICI 182,780 (an anti-estrogen) blocked TRPV6 expression via estrogen receptors in mice. The juxtaposition of uterine and placental TRPV6 expressed in these tissues supports the notion that TRPV6 participates in transferring calcium ions between the maternal and fetal compartments. Taken together, TRPV6 gene may function as a key element in controlling calcium transport in the uterus between the embryo and the placenta during pregnancy.