Reactive Oxygen Species Downregulate Transient Receptor Potential Melastatin 6 Expression Mediated by the Elevation of miR-24-3p in Renal Tubular Epithelial Cells.

BACKGROUND: A low level of serum magnesium ion (Mg2+) is associated with type 2 diabetes mellitus (T2D). However, the molecular mechanism of Mg2+ deficiency has not been fully clarified. The current study sought to assesses the effect of reactive oxygen species on the expression of Mg2+ channels and miRNA. METHODS: The expression of Mg2+ channels and miRNA were examined by real-time polymerase chain reaction. Intracellular Mg2+ concentration was measured by Magnesium Green fluorescence measurement. RESULTS: The mRNA level of transient receptor potential melastatin 6 (TRPM6), which functions as Mg2+ influx channel in the distal convoluted tubule (DCT) of the kidney, was decreased by glycated albumin (GA), but not by insulin in rat renal tubule-derived NRK-52E cells. The mRNA levels of TRPM7, a homologue of TRPM6, and CNNM2, a Mg2+ efflux transporter located at the basolateral membrane of DCT, were changed by neither GA nor insulin. The generation of reactive oxygen species (ROS) was increased by GA. Hydrogen peroxide (H2O2) dose-dependently decreased TRPM6 mRNA, but it inversely increased the reporter activity of TRPM6. H2O2 accelerated the degradation of TRPM6 mRNA in actinomycin D assay without affecting TRPM7 and CNNM2 mRNA expressions. Nine miRNAs were considered as candidates for the regulator of stability of TRPM6 mRNA. Among them, miR-24-3p expression was increased by H2O2. The H2O2-induced reduction of TRPM6 mRNA was rescued by miR-24-3p siRNA. Magnesium Green fluorescence measurement showed that Mg2+ influx is suppressed by H2O2, which was rescued by an antioxidant and miR-24-3p siRNA. CONCLUSIONS: We suggest that GA decreases TRPM6 expression mediated by the elevation of ROS and miR-24-3p in renal tubular epithelial cells of T2D.

Elevation of Chemosensitivity of Lung Adenocarcinoma A549 Spheroid Cells by Claudin-2 Knockdown through Activation of Glucose Transport and Inhibition of Nrf2 Signal.

Claudin-2 (CLDN2), a tight junctional protein, is involved in the chemoresistance in a three-dimensional spheroid culture model of human lung adenocarcinoma A549 cells. However, the mechanism has not been fully clarified. We found that the knockdown of CLDN2 expression by siRNA in the spheroid reduces the expression of glucose transporters and metabolic enzymes. In a two-dimensional culture model, the expression of these proteins was increased by glucose deprivation or fasentin, an inhibitor of glucose transporter. In addition, the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant enzymes including heme oxygenase-1, NAD(P)H:quinone oxidoreductase-1, and a glutamate-cysteine ligase modifier subunit were increased by fasentin. The fluorescence intensities of JC-1, a probe of mitochondrial membrane potential, and MitoROS 580, a probe of mitochondrial superoxide production, were increased by fasentin. These results suggest that mitochondrial production of reactive oxygen species is increased by glucose deficiency. The knockdown of CLDN2 enhanced the flux of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), a fluorescent deoxyglucose derivative, in a transwell assay, and the accumulation of glucose and 2-NBDG in spheroid cells. The expression of Nrf2 was decreased by CLDN2 knockdown, which was inhibited by fasentin and sulforaphane, a typical Nrf2 activator, in spheroid cells. The sensitivity of spheroid cells to doxorubicin, an anthracycline antitumor antibiotic, was enhanced by CLDN2 knockdown, which was inhibited by fasentin and sulforaphane. We suggest that CLDN2 induces chemoresistance in spheroid cells mediated through the inhibition of glucose transport and activation of the Nrf2 signal.

Inverse regulation of claudin-2 and -7 expression by p53 and hepatocyte nuclear factor 4α in colonic MCE301 cells.

Colonic epithelial cells move up along the crypt villus axis and are differentiated into absorptive or secretory cells. Claudin-7 (CLDN7), a tight junctional protein, is mainly located at the surface of crypt, whereas CLDN2 is located at the bottom. However, the expression mechanism and function of these CLDNs are not fully understood. The expression levels of CLDN2 and CLDN7 were altered depending on the culture days in MCE301 cells derived from mouse colon. The nuclear levels of transcriptional factors p53 and hepatocyte nuclear factor 4α (HNF4α) at day 21 were higher than those at day 7. Tenovin-1 (TEN), a p53 activator, increased the nuclear levels of p53 and HNF4α. The mRNA level and promoter activity of CLDN7 were increased by TEN, whereas those of CLDN2 were decreased. The changes of CLDNs expression were inhibited by p53 and HNF4α siRNAs. The association between p53 and HNF4α was elevated by TEN. In addition, the binding of p53 and HNF4α to the promoter region of CLDN2 and CLDN7 was enhanced by TEN. Transepithelial electrical resistance was decreased by TEN, but paracellular fluxes of lucifer yellow and dextran were not. In the Ussing chamber assay, TEN increased dilution potential and the ratio of permeability of Cl- to Na+. Both p53 and HNF4α were highly expressed at the surface of mouse colon crypt. We suggest that p53 and HNF4α alter the paracellular permeability of Cl- to Na+ mediated by the inverse regulation of CLDN2 and CLDN7 expression in the colon.

Increase in Toxicity of Anticancer Drugs by PMTPV, a Claudin-1-Binding Peptide, Mediated via Down-Regulation of Claudin-1 in Human Lung Adenocarcinoma A549 Cells.

Claudin-1 (CLDN1), a tight junctional protein, is highly expressed in lung cancer cells and may contribute to chemoresistance. A drug which decreases CLDN1 expression could be a chemosensitizer for enhancing the efficacy of anticancer drugs, but there is no such drug known. We found that PMTPV, a short peptide, which mimics the structure of second extracellular loop (ECL2) of CLDN1, can reduce the protein level of CLDN1 without affecting the mRNA level in A549 cells derived from human lung adenocarcinoma. The PMTPV-induced decrease in CLDN1 expression was inhibited by monodansylcadaverine, a clathrin-mediated endocytosis inhibitor, and chloroquine, a lysosome inhibitor. Quartz crystal microbalance assay showed that PMTPV can directly bind to the ECL2 of CLDN1. In transwell assay, PMTPV increased fluxes of Lucifer yellow (LY), a paracellular flux marker, and doxorubicin (DXR), an anthracycline anticancer drug, without affecting transepithelial electrical resistance. In three-dimensional spheroid culture, the size and cell viability were unchanged by short peptides, but the fluorescence intensity of hypoxia probe LOX-1 was decreased by PMTPV. PMTPV elevated the accumulation and cytotoxicity of DXR in the spheroids. Similar results were observed by knockdown of CLDN1. Furthermore, the sensitivities to cisplatin (CDDP), docetaxel, and gefitinib were enhanced by PMTPV. The level of CLDN1 expression in CDDP-resistant cells was higher than that in parental A549 cells, which was reduced by PMTPV. PMTPV restored the toxicity to DXR in the CDDP-resistant cells. Our data suggest that PMTPV may become a novel chemosensitizer for lung adenocarcinoma.

Upregulation of Claudin-7 Expression by Angiotensin II in Colonic Epithelial Cells of Mice Fed with NaCl-Depleted Diets.

Dietary NaCl depletion increases Na+ and Cl- absorption in the colon, but the mechanisms are not fully understood. So far, we reported that the expression of claudin-7 (CLDN7), a tight junction (TJ) protein, was upregulated in the mice fed with NaCl-depleted diets, but the regulatory mechanism has not been clarified. Here, we found that angiotensin II (ANGII) increases the mRNA level of CLDN7, which was inhibited by losartan, a type 1 ANGII (AT1) receptor antagonist. Immunofluorescence measurement showed that CLDN7 is colocalized with zonula occludens-1 at the TJ in untreated and ANGII-treated cells. ANGII decreased transepithelial electrical resistance (TER) and increased permeability to C1- without affecting permeability to lucifer yellow, a paracellular flux marker. In contrast, TER was increased by CLDN7 knockdown in the absence and presence of ANGII. ANGII increased the nuclear distribution of phosphorylated p65 subunit of NF-κB, which was inhibited by losartan. The ANGII-induced elevation of CLDN7 expression was blocked by BAY 11-7082 (BAY), an NF-κB inhibitor. Luciferase reporter assay showed that ANGII increases promoter activity of CLDN7, which was inhibited by the treatment with losartan or BAY, and introduction of mutations in κB-binding motifs in the promoter. The binding of p65 on the promoter region of CLDN7 was increased by ANGII, which was inhibited by losartan and BAY in chromatin immunoprecipitation assay. Our data suggest that ANGII acts on AT1 receptor and increases paracellular permeability to Cl- mediated by the elevation of CLDN7 expression in the colon.

Claudin-2 binding peptides, VPDSM and DSMKF, down-regulate claudin-2 expression and anticancer resistance in human lung adenocarcinoma A549 cells.

Claudin-2 (CLDN2), a tight junctional protein, is involved in the chemoresistance in spheroid culture models of human lung adenocarcinoma A549 cells. However, there is no chemical which can improve the sensitivity to anticancer drugs. So far, we reported that DFYSP, a short peptide which mimics the second extracellular loop (ECL2) of CLDN2, decreases CLDN2 expression in A549 cells, but the concentration is relatively high. Here, we found that the effects of VPDSM and DSMKF are stronger than that of DFYSP. Both VPDSM and DSMKF decreased the protein levels of CLDN2 without affecting the mRNA levels of CLDN2. The peptide-induced decrease in CLDN2 expression was suppressed by monodansylcadaverine (MDC), a clathrin-dependent endocytosis (CDE) inhibitor, and chloroquine, a lysosome inhibitor. CLDN2 was colocalized with ZO-1, an adapter protein, in tight junctions (TJs) under control conditions, whereas it disappeared from the TJs in the peptide-treated cells. Quartz crystal microbalance assay showed that both peptides can bind to recombinant CLDN2 protein. Both peptides increased permeability to paracellular transport marker lucifer yellow. In three-dimensional spheroid culture models, both peptides enhanced the sensitivity to doxorubicin, a cytotoxic anticancer drug, which was inhibited by MDC. We suggest that VPDSM and DSMKF enhance the chemosensitivity to anticancer drugs in aggregated adenocarcinoma cells mediated by the CDE pathway and lysosomal degradation of CLDN2 in lung adenocarcinoma cells. VPDSM and DSMKF, which mimic the ECL2 of CLDN2, may become novel adjuvant therapeutic drugs for lung adenocarcinoma.

Cyanidin Increases the Expression of Mg2+ Transport Carriers Mediated by the Activation of PPARα in Colonic Epithelial MCE301 Cells.

Mg2+ deficiency may be involved in lifestyle-related diseases, including hypertension, cardiovascular diseases, and diabetes mellitus. Dietary Mg2+ is absorbed in the intestine mediated through transcellular and paracellular pathways. However, there is little research into what factors upregulate Mg2+ absorption. We searched for food constituents that can increase the expression levels of Mg2+ transport carriers using mouse colonic epithelial MCE301 cells. Cyanidin, an anthocyanidin found in black beans and berries, increased the mRNA levels of Mg2+ transport carriers including transient receptor potential melastatin 6 (TRPM6) channel and cyclin M4 (CNNM4). The cyanidin-induced elevation of Mg2+ transport carriers was blocked by GW6471, a peroxisome proliferator-activated receptor α (PPARα) inhibitor, but not by PPARγ, PPARδ, and protein kinase A inhibitors. Cyanidin-3-glucoside showed similar results to cyanidin. Cyanidin increased the protein levels of TRPM6 and CNNM4, which were distributed in the apical and lateral membranes, respectively. The nuclear localization of PPARα and reporter activities of Mg2+ transport carriers were increased by cyanidin, which were inhibited by GW6471. The cyanidin-induced elevation of reporter activity was suppressed by a mutation in a PPAR-response element. Fluorescence measurements using KMG-20, an Mg2+ indicator, showed that Mg2+ influx and efflux from the cells were enhanced by cyanidin, and which were inhibited by GW6471. Furthermore, cyanidin increased paracellular Mg2+ flux without affecting transepithelial electrical resistance. We suggest that cyanidin increases intestinal Mg2+ absorption mediated by the elevation of TRPM6 and CNNM4 expression, and may constitute a phytochemical that can improve Mg2+ deficiency.

Sodium Citrate Increases Expression and Flux of Mg2+ Transport Carriers Mediated by Activation of MEK/ERK/c-Fos Pathway in Renal Tubular Epithelial Cells.

A chronic magnesium deficiency may be one of the causes of lifestyle-related diseases such as hypertension and diabetes. Serum Mg2+ concentration is strictly controlled by the reabsorption pathway in the renal tubules, but little is known about how Mg2+ reabsorption is upregulated. We searched for food compounds which can increase the expression levels of Mg2+ transport carriers including transient receptor potential melastatin 6 (TRPM6) channel and cyclin M2 (CNNM2). Sodium citrate (SC) increased the mRNA levels of TRPM6 and CNNM2 in renal tubular epithelial NRK-52E cells. The SC-induced elevation of TRPM6 was inhibited by U0126, a mitogen-activated protein kinase kinase (MEK) inhibitor, but the CNNM2 was not. SC increased the levels of p-ERK1/2 and p-c-Fos, which were inhibited by U0126. SC induced alkalization of culture medium. Both SC and alkalization enhanced Mg2+ influx, which was inhibited by U0126 and introduction of TRPM6 siRNA. The reporter activity of TRPM6 was increased by SC and alkalization, which was suppressed by mutation in an AP-1-binding site. The SC-induced elevation of p-ERK1/2 and p-EGFR was inhibited by diphenylene iodonium, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, and erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. SC did not change the level of acetyl histone H3, but increased the association of c-Fos with the promoter region of TRPM6. These results suggest that SC increases TRPM6 expression and Mg2+ influx mediated by the activation of NADPH oxidase and an EGFR/ERK/c-Fos pathway in the renal tubules.