TRPV6 Research Articles

Novel TRPV6 variant linked with transient neonatal hyperparathyroidism

BackgroundHereditary transient neonatal hyperparathyroidism (TNHP) is a rare autosomal-recessive condition caused by variants in TRPV6 gene which encodes for a transient maternal–fetal calcium transport channel. This is characterized by interference with placental maternal–fetal calcium transport causing fetal calcium deficiency. It primarily manifests as defective bone mineralization, narrow and bell-shaped thorax, bone fractures and short bones at birth. The current study aimed to describe a novel TRPV6 variant linked with TNHP in an Indian family and the review of literature.Case presentationThe proband is a term female neonate with fetal growth restriction born to a third-degree consanguineous couple. She was noted to have diffuse defective bone mineralization, narrow and bell-shaped thoracic cavity, short bones and curved ribs without any bone fractures. The first pregnancy was affected with similar features in the fetus and had been terminated. Parental whole-exome sequencing suggested heterozygous missense variant in exon 12 of the TRPV6 gene (c.1585G > A, p.Asp529Asn) in both the parents. The proband required non-invasive respiratory support for ten days in neonatal intensive care unit. She had low calcium and high parathyroid hormone (PTH) and alkaline phosphatase (ALP) levels. She received calcium, phosphorus and vitamin D supplements for three months leading to normalization of serum PTH and ALP levels. Whole-exome sequencing of the proband suggested a homozygous missense variant in exon 12 of the TRPV6 gene (p.Asp529Asn; ENST00000359396.9) that results in the amino acid substitution of asparagine for aspartic acid at codon 529. To the best of author’s knowledge, this is the twelfth case of TNHP reported in literature. The novel variant of TRPV6 gene present in this family has not been reported earlier.ConclusionOur finding broadens the genotypic spectrum of TNHP.

Diagnostic yield of repeat genetic testing in idiopathic chronic pancreatitis

Genetic testing is performed for unexplained pancreatitis. The aim of this study was to evaluate the diagnostic value of repeating genetic testing in idiopathic pancreatitis when new predisposing genes are identified.We investigated 330 patients who were initially screened for PRSS1, SPINK1 and CFTR genes. A new analysis was performed by Next-Generation Sequencing (NGS) for PRSS1, SPINK1, CFTR, CTRC, CASR, CPA1, TRPV6 genes and the CEL-HYB1 allele in clinical practice, and patients were included in our cohort study. Additional rare variants were identified in 7.3 % of the patients.Screening for new pancreatitis genes is recommended when initial screening is limited. Routine use of NGS is a useful diagnostic tool in these cases.

Effect of Vitamin D overdose on VDR, TRPV6 and CYP3A11 genes expression, biochemical tests and histopathological legions in Albino mice

Effect of Vitamin D overdose on VDR, TRPV6 and CYP3A11 genes expression, biochemical tests and histopathological legions in Albino mice

Characterization of the TRPV6 calcium channel-specific phenotype by RNA-seq in castration-resistant human prostate cancer cells.

Background: Transient receptor potential vanilloid subfamily member 6 (TRPV6), a highly calcium-selective channel, has been shown to play a significant role in calcium homeostasis and to participate both in vitro and in vivo in growth, cell survival, and drug resistance of prostate cancer. Its role and the corresponding calcium-dependent pathways were mainly studied in hormone-dependent human prostate cancer cell lines, often used as a model of early-stage prostate cancers. The goal of the present study was to describe the TRPV6-specific phenotype and signaling pathways it is involved in, using castration-resistant prostate cancer cell lines. Methods: RNA sequencing (RNA-seq) was used to study the gene expression impacted by TRPV6 using PC3Mtrpv6-/- versus PC3Mtrpv6+/+ and its derivative PC3M-luc-C6trpv6+/+ cell line in its native and TRPV6 overexpressed form. In addition to the whole-cell RNA sequencing, immunoblotting, quantitative PCR, and calcium imaging were used to validate trpv6 gene status and functional consequences, in both trpv6 -/- and TRPV6 overexpression cell lines. Results: trpv6 -/- status was validated using both immunoblotting and quantitative PCR, and the functional consequences of either trpv6 gene deletion or TRPV6 overexpression were shown using calcium imaging. RNA-seq analysis demonstrated that the calcium channel TRPV6, being a crucial player of calcium signaling, significantly impacts the expression of genes involved in cancer progression, such as cell cycle regulation, chemotaxis, migration, invasion, apoptosis, ferroptosis as well as drug resistance, and extracellular matrix (ECM) re-organization. Conclusion: Our data suggest that the trpv6 gene is involved in and regulates multiple pathways related to tumor progression and drug resistance in castration-resistant prostate cancer cells.

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.

Mutations That Affect the Surface Expression of TRPV6 Are Associated with the Upregulation of Serine Proteases in the Placenta of an Infant.

Recently, we reported a case of an infant with neonatal severe under-mineralizing skeletal dysplasia caused by mutations within both alleles of the TRPV6 gene. One mutation results in an in frame stop codon (R510stop) that leads to a truncated, nonfunctional TRPV6 channel, and the second in a point mutation (G660R) that, surprisingly, does not affect the Ca2+ permeability of TRPV6. We mimicked the subunit composition of the unaffected heterozygous parent and child by coexpressing the TRPV6 G660R and R510stop mutants and combinations with wild type TRPV6. We show that both the G660R and R510stop mutant subunits are expressed and result in decreased calcium uptake, which is the result of the reduced abundancy of functional TRPV6 channels within the plasma membrane. We compared the proteomic profiles of a healthy placenta with that of the diseased infant and detected, exclusively in the latter two proteases, HTRA1 and cathepsin G. Our results implicate that the combination of the two mutant TRPV6 subunits, which are expressed in the placenta of the diseased child, is responsible for the decreased calcium uptake, which could explain the skeletal dysplasia. In addition, placental calcium deficiency also appears to be associated with an increase in the expression of proteases.

Preparation of casein phosphopeptides calcium complex and the promotion in calcium cellular uptake through transcellular transport pathway.

This study evaluated the stability of casein phosphopeptides (CPP) and obtained peptide-calcium complex by heating and chelating the peptide with CaCl2 in a neutral solution. To assess the bioavailability of various calcium formulations, the calcium transport models were established in Caco-2 cells, and the transcellular transport pathways of various calcium formulations were studied by RT-PCR and Western blotting. Results of circular dichroism showed that CPP was a stable polypeptide. The ultraviolet absorption spectrum and Fourier transform-infrared spectrum (FT-IR) indicated that calcium could be chelated by carboxyl oxygen and amino nitrogen atoms of CPP to form peptide calcium chelate, and the calcium bioavailability of peptide calcium chelate was significantly higher than that of CaCl2 , calcium l-aspartate, and casein phosphopeptides mixed with CaCl2 . Four calcium sources increased the expression of TRPV5 and TRPV6 genes and proteins. The study intended to provide a basis for developing a novel calcium supplement. PRACTICAL APPLICATIONS: This paper examined the bioavailability of casein phosphopeptides calcium complex, CaCl2 , calcium l-aspartate, and casein phosphopeptides mixed with CaCl2 in Caco-2 cells, and the mechanisms were detected by western blotting. The results provide theoretical knowledge for the selection of calcium supplement raw materials and lay a foundation for the development of compound calcium preparations and drugs in the future.

Expression of transient receptor potential vanilloid genes and proteins in diabetic rat heart.

Cardiac complications are leading causes of death in diabetic patients. Imbalance of Ca2+ homeostasis is a hallmark of cardiac dysfunction in diabetes, while TRPV channels are non-selective for cations and are permeable to Ca2+. Our aim was to evaluate the expression levels of TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, and TRPV6 genes and proteins in cardiac tissue at 3 days and 4, 8, and 12 weeks after induction of diabetes. Sprague-Dawley rats were assigned to control and DM groups. DM was induced by intraperitoneal injection of streptozotocin (60mg/kg). The expression levels of TRPV genes were analyzed by the quantitative reverse transcription polymerase chain reaction, and TRPV proteins were determined by western blotting. Compared to controls, the expression levels of TRPV2, TRPV3, and TRPV6 in diabetic myocardium did not change, while TRPV1 decreased at 4, 8, and 12 weeks, TRPV4 was upregulated at 3 days and 4, 8, and 12 weeks, TRPV5 mRNA increased at 8 and 12 weeks, and TRPV5 protein increased at 4, 8, and 12 weeks. Our findings showed that TRPV1, TRPV4, and TRPV5 are associated with the diabetic heart.

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.

Association between histone deacetylase activity and vitamin D-dependent gene expressions in relation to sulforaphane in human colorectal cancer cells.

It is relatively unknown as to how dietary bioactive compound sulforaphane (SFN) and vitamin D regulate gene expression in colorectal cancer. We hypothesized that a combination of SFN with vitamin D would prove beneficial in colorectal cancer. A combinatorial chemo-preventive strategy was employed to investigate the impact of SFN on chromatin remodeling in colorectal carcinoma. To understand the epigenetics-mediated changes in gene expression in response to SFN and vitamin D, Caco-2 cells were exposed for 24 h to vitamin D (100 nmol L-1 ) either alone or in combination with SFN and trichostatin A (20 and 1 μmol L-1 , respectively) at 70% confluency (proliferating) and after 13 days post-confluency (fully differentiated). Changes to VDR, CYP24A1, CYP27B1 and TRPV6 gene expressions were quantified using real-time PCR-based assays. Histone deacetylase (HDAC) inhibitor activity was assessed using HDAC I/II assay that measured global changes in acetylation status. In differentiated Caco-2 cells, none of the genes had significant changes from D alone group. D + SFN (P = 0.99) demonstrated an opposing effect from D alone and decreased VDR expression. However, in proliferating Caco-2 cells, D + SFN (P < 0.04) increased VDR expression and decreased CYP27B1 (P < 0.01) more than D alone (P = 0.38 and 0.07, respectively). Although statistically significant, D + SFN (P = 0.01) effect on HDAC inhibitor activity was less than trichostatin A alone group (P < 0.0004) or SFN alone group (P < 0.0014). The data suggest that colon cancer cells respond to dietary components differently under different conditions. The effect of vitamin D and SFN is selective and gene-specific in the complex multistep process of colorectal carcinogenesis in vitro. © 2020 Society of Chemical Industry.

Why endogenous TRPV6 currents are not detectable-what can we learn from bats?

TRPV6 is a calcium selective TRP channel and is expressed in many species. TRPV6 transcripts are abundantly expressed in few tissues but strangely channel properties are only accessible to electrophysiological recordings after overexpression whereas in native tissue functional channel currents seem not to be detectable. Another exceptional property of human and mouse TRPV6 proteins is that the initiation of translation starts from a non-canonical ACG triplet which is translated as methionine. This triplet is located 120 bp upstream of the first in-frame AUG codon of the human/mouse TRPV6 mRNA. In contrast, the TRPV6 gene of bats is initiated from an AUG triplet at the corresponding position of the human ACG. On the basis of these structural nucleotide differences between human and bats we studied the role of the absolute N-Terminus for the regulation of translation by developing chimera and mutants of human/bat TRPV6 channels. The human sequence which is located downstream of the initiation codon slows down ribosomal scanning in 3′ direction. We suggest that the mechanism involves most likely the deceleration of ribosome scanning by stem-loop formation and the use of the common initiator tRNA, tRNAiMet, which is placed onto the inappropriate ACG codon resulting in low protein synthesis. The reduced translation efficiency is important to protect TRPV6 expressing cells from toxic calcium overload. The regulation of the TRPV6 translation in bats may be an adaptation to low calcium amounts present in the natural nutrition. In addition, we show that also the GFP protein can be controlled using the translational mechanism of human TRPV6.

Modulation of Calcium Homeostasis May Be Associated with Susceptibility to Renal Cell Carcinoma in Diabetic Nephropathy Rats.

IntroductionClinical studies have indicated a relationship between diabetic nephropathy (DN) and the incidence and prevalence of renal cell carcinoma (RCC). However, the mechanism linking diabetic nephropathy and renal cell carcinoma has not yet to be identified.MethodsIn this study, a total of 42 male Sprague Dawley (SD) rats were randomly assigned to a DN group (n=35) and a control group (n=7). All animals in the DN group were unilaterally nephrectomized and treated with streptozotocin with the development of blood glucose levels >16.7mmol/L and dominant proteinuria and were compared to controls without such changes. Histopathologic alterations in the kidneys were examined by HE staining and Ki-67 immunohistochemistry. Differentially expressed genes were identified and validated by RNA-seq and PCR.ResultsAs the results, except for two rats that failed to develop the DN model and were excluded from the analysis, 33 rats in the DN group with overt signs of DN demonstrated significantly higher food and water intake, urine production, and urine protein and urinary protein/creatinine ratio than controls. Overall, 15.2% (n=5/33) of DN animals developed RCC while none tumors were observed in the control group (n=0/7). RNA-seq analysis in these animals indicated different TRPV5 gene expression and calcium pathway expression in DN animals with developing tumors, when compared with animals with no obvious tumors. In addition, DN animals diagnosed with RCC showed increased expression of GLUT2 and c-met, when compared to controls and DN animals without tumors.DiscussionIn conclusion, the disordered calcium metabolism, especially disturbed TRPV5 mediated Ca2+ signal, may have been related to the development of RCC in DN rats. Further studies related to the detailed mechanism are still needed.

Dexamethasone Treatment Increases the Intracellular Calcium Level Through TRPV6 in A549 Cells.

This study investigated the effect of dexamethasone (DEX) on intracellular calcium levels and the expressions of transient receptor potential cation channel subcomponent V member 6 (TRPV6), sodium-calcium exchanger 1 (NCX1), and plasma membrane calcium ATPase 1 (PMCA1) in A549 cells. The intracellular calcium level, by using the calcium indicator pGP-CMV-GCaMP6f, increased following DEX treatment for 6, 12, and 24 h in A549 cells. In addition, Rhod-4 assay after DEX treatment for 24 h showed that DEX increased the level of intracellular calcium. The expression of the calcium influx TRPV6 gene significantly increased, whereas the expressions of the calcium outflow NCX1 and PMCA1 genes significantly decreased with DEX treatment. The mRNA levels of surfactant protein genes SFTPA1, SFTPB, SFTPC, and SFTPD and the secreted airway mucin genes MUC1 and MUC5AC were investigated by treating cells with DEX. The DEX treatment decreased the mRNA levels of SFTPA1 and SFTPB but increased the mRNA levels of SFTPC and SFTPD. The MUC1 mRNA level was increased by DEX treatment, whereas MUC5AC mRNA was significantly decreased. These results indicate that DEX influences the intracellular calcium level through TRPV6, and affects pulmonary surfactant genes and secreted airway mucin genes in A549 cells.

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.

Histone deacetylase activity and vitamin D-dependent gene expressions in relation to sulforaphane in human breast cancer cells.

This study employed a combinatorial chemopreventive strategy to investigate the impact of dietary histone deacetylase (HDAC) inhibitor, that is, sulforaphane on chromatin remodeling in BC. To understand the epigenetics-mediated changes in gene expression, MCF-7 cells were exposed for 24hr to 1,25(OH)2D3 (100nM) either alone or in combination with L-sulforaphane and TSA (20μM and 1μM, respectively) at 70% confluency. Changes in VDR, CYP24A1, CYP27B1, and TRPV6 gene expressions were quantified using real-time PCR-based assays. HDAC inhibitor activity was assessed using HDAC I/II assay that measured global changes in acetylation status. Cell viability was measured using ATP and MT assays. Clonogenic and migration assays were performed to analyze the ability of single cells to grow into colonies and % closure (migration ability) upon treatments, respectively. Results were expressed as ΔCT±standard error of means (SEM) from One-way ANOVA analyses for mRNA expressions and mean±SEM for all other assays. In MCF-7 cells, treatment with 1,25(OH)2D3 tended to decrease VDR (13±0.4) and CYP27B1 (12±0.96), while significantly increased TRPV6 (p=.02, 14±0.1) and CYP24A1 (p<.0001, 0.38±0.12) expression. D alone and D+TSA group had the opposite effects on HDAC inhibition from SFN alone, D+SFN, and TSA alone. The clonogenic assay showed a significant decrease in colony formation with no colonies for D+TSA (p<.03) and TSA alone groups (p<.03). Cell viability tended to decrease with D alone and in combination with TSA. These data suggest that the effects of 1,25(OH)2D3 and sulforaphane are selective and gene-specific in MCF-7 cells. Breast cancer (BC) affects a large number of the U.S. population each year. Like most cancers, nutrition does play a role in the prevention of BC. However, dietary advice that includes reducing alcohol intake, red meat, and saturated fat consumption, while increasing the intake of heart-healthy fats, dietary fiber, and lean protein, etc., is difficult to apply to all cancers from a preventative standpoint. Vitamin D has been implicated in BC, mostly as a protective factor, with mixed findings. This research focuses on the role of vitamin D as a protective intervention in human BC, along with a dietary bioactive compound-sulforaphane. The idea is to combine the known benefits of a micronutrient with potential benefits of the bioactive compound to establish a stronger intervention against BC progression, irrespective of the subtype.

Association of TRPV5 gene polymorphism with calcium urolithiasis: a case–control study from West Bengal, India

Present study was intended to investigate the potential contribution of TRPV5 gene polymorphisms with calcium urolithiasis in the population of West Bengal, India. A case-control study was performed with 152 calcium urolithiasis patients and 144 corresponding healthy controls. Epidemiological and clinical parameters were documented as well as peripheral blood sample was collected from each individual, followed by genomic DNA isolation. Then to identify genetic variants of TRPV5, the entire coding region and exon-intron boundaries of the gene were amplified by polymerase chain reaction using specific oligonucleotide primers and then genotypes were determined by bi-directional DNA sequencing and sequence alignment between case and control individuals. Urinary calcium excretion was found to be significantly high (p value < 0.0001) in urolithiasis patients as compared to controls. A total of 14 SNPs were obtained of which one non-synonymous (rs4236480; p.Arg154His; CGT > CAT), one synonymous (rs4252417; p.Tyr278Tyr; TAC > TAT) and three intronic (rs4252400, rs4252402, rs4236481) SNPs were found to be significantly associated with increased risk of urolithiasis. For non-synonymous SNP rs4236480, 'A' was found to be the risk allele (OR 1.77, 95% CI 1.24-2.51; p value 0.001) and genotype frequency analysis revealed that individuals carrying variant genotype AA were more prone to the disease than individuals with wild genotype GG (OR 3.09, 95% CI 1.26-7.59; p value 0.0136), indicating AA as the risk genotype. The non-synonymous SNP rs4236480 showed significant association with urolithiasis risk in West Bengal population of India. Future translational and larger population-based studies are required to validate our finding.

Regulatory effect of dexamethasone on tracheal calcium processing proteins and mucosal secretion.

Dexamethasone inhibits mucin secretion considering the primary option for treating acute asthma exacerbation. However, the mechanism underlying dexamethasone-induced decreased in mucosecretion is unclear. Recent studies have reported that dexamethasone exerts an inhibitory effect on mucosecretion in the lung by modulating the expression of calcium processing genes. However, the expression of the calcium processing genes in the trachea is not examined yet. Thus, the present study is the first to report the localization of calcium processing proteins such as transient receptor potential vanilloid-4 (Trpv4), transient receptor potential vanilloid-6 (Trpv6), calbindin-D9k (CaBP-9k) and plasma membrane Ca2+-ATPase 1 (Pmca1) in the mouse trachea and their glucocorticoid-induced response. In this study, mice were subcutaneously injected with dexamethasone for 5 days, and their tracheal samples were collected by dividing the trachea into the cervical, and thoracic sections based on its anatomical structure. The localization of TRPV4, TRPV6, CaBP-9k, and PMCA1 proteins was detected in the tracheal epithelium, submucosal glands, cartilages and muscles. Dexamethasone treatment downregulated the mRNA expression of the four calcium processing genes and mucin producing genes. The dexamethasone-induced decrease in the secretion of mucosubstances in the trachea was determined by performing Alcian blue-periodic acid-Schiff staining. Thus, the findings of the present study suggest that glucocorticoids simultaneously can regulate the expression of calcium processing genes and tracheal mucosecretion.

Modeling the structural and dynamical changes of the epithelial calcium channel TRPV5 caused by the A563T variation based on the structure of TRPV6

TRPV5, transient receptor potential cation channel vanilloid subfamily member 5, is an epithelial Ca2+ channel that plays a key role in the active Ca2+ reabsorption process in the kidney. A single nucleotide polymorphism (SNP) rs4252499 in the TRPV5 gene results in an A563T variation in the sixth transmembrane (TM) domain of TRPV5. Our previous study indicated that this variation increases the Ca2+ transport function of TRPV5. To understand the molecular mechanism, a model of TRPV5 was established based on the newly deposited structure of TRPV6 that has 83.1% amino acid identity with TRPV5 in the modeled region. Computational simulations were performed to study the structural and dynamical differences between the TRPV5 variants with A563 and T563. Consistent with the TRPV1-based simulation, the results indicate that the A563T variation increases the contacts between residues 563 and V540, which is one residue away from the key residue D542 in the Ca2+-selective filter. The variation enhanced the stability of the secondary structure of the pore region, decreased the fluctuation of residues around residue 563, and reduced correlated and anti-correlated motion between monomers. Furthermore, the variation increases the pore radius at the selective filter. These findings were confirmed using simulations based on the recently determined structure of rabbit TRPV5. The simulation results provide an explanation for the observation of enhanced Ca2+ influx in TRPV5 caused by the A563T variation. The A563T variation is an interesting example of how a residue distant from the Ca2+-selective filter influences the Ca2+ transport function of the TRPV5 channel.Communicated by Ramaswamy H. Sarma

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.