Decreased Membrane Expression Research Articles

Helicobacter pylori-Induced Decrease in Membrane Expression of Na,K-ATPase Leads to Gastric Injury.

Helicobacter pylori is a highly prevalent human gastric pathogen that causes gastritis, ulcer disease, and gastric cancer. It is not yet fully understood how H. pylori injures the gastric epithelium. The Na,K-ATPase, an essential transporter found in virtually all mammalian cells, has been shown to be important for maintaining the barrier function of lung and kidney epithelia. H. pylori decreases levels of Na,K-ATPase in the plasma membrane of gastric epithelial cells, and the aim of this study was to demonstrate that this reduction led to gastric injury by impairing the epithelial barrier. Similar to H. pylori infection, the inhibition of Na,K-ATPase with ouabain decreased transepithelial electrical resistance and increased paracellular permeability in cell monolayers of human gastric cultured cells, 2D human gastric organoids, and gastric epithelium isolated from gerbils. Similar effects were caused by a partial shRNA silencing of Na,K-ATPase in human gastric organoids. Both H. pylori infection and ouabain exposure disrupted organization of adherens junctions in human gastric epithelia as demonstrated by E-cadherin immunofluorescence. Functional and structural impairment of epithelial integrity with a decrease in Na,K-ATPase amount or activity provides evidence that the H. pylori-induced downregulation of Na,K-ATPase plays a role in the complex mechanism of gastric disease induced by the bacteria.

Alterations in HCN1 expression and distribution during epileptogenesis in rats

BackgroundThe hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN1) is predominantly located in key regions associated with epilepsy, such as the neocortex and hippocampus. Under normal physiological conditions, HCN1 plays a crucial role in the excitatory and inhibitory regulation of neuronal networks. In temporal lobe epilepsy, the expression of HCN1 is decreased in the hippocampi of both animal models and patients. However, whether HCN1 expression changes during epileptogenesis preceding spontaneous seizures remains unclear. ObjectiveThe aim of this study was to determine whether the expression of HCN1 is altered during the epileptic prodromal phase, thereby providing evidence for its role in epileptogenesis. MethodsWe utilized a cobalt wire-induced rat epilepsy model to observe changes in HCN1 during epileptogenesis and epilepsy. Additionally, we also compared HCN1 alterations in epileptogenic tissues between cobalt wire- and pilocarpine-induced epilepsy rat models. Long-term video EEG recordings were used to confirm seizures development. Transcriptional changes, translation, and distribution of HCN1 were assessed using high-throughput transcriptome sequencing, total protein extraction, membrane and cytoplasmic protein fractionation, western blotting, immunohistochemistry, and immunofluorescence techniques. ResultsIn the cobalt wire-induced rat epilepsy model during the epileptogenesis phase, total HCN1 mRNA and protein levels were downregulated. Specifically, the membrane expression of HCN1 was decreased, whereas cytoplasmic HCN1 expression showed no significant change. The distribution of HCN1 in the distal dendrites of neurons decreased. During the epilepsy period, similar HCN1 alterations were observed in the neocortex of rats with cobalt wire-induced epilepsy and hippocampus of rats with lithium pilocarpine-induced epilepsy, including downregulation of mRNA levels, decreased total protein expression, decreased membrane expression, and decreased distal dendrite expression. ConclusionsAlterations in HCN1 expression and distribution are involved in epileptogenesis beyond their association with seizure occurrence. Similarities in HCN1 alterations observed in epileptogenesis-related tissues from different models suggest a shared pathophysiological pathway in epileptogenesis involving HCN1 dysregulation. Therefore, the upregulation of HCN1 expression in neurons, maintenance of the HCN1 membrane, and distal dendrite distribution in neurons may represent promising disease-modifying strategies in epilepsy.

Targeted transcriptional upregulation of SENP1 by CRISPR activation enhances deSUMOylation pathways to elicit antinociception in the spinal nerve ligation model of neuropathic pain.

The voltage-gated sodium channel Na V 1.7 is an essential component of human pain signaling. Changes in Na V 1.7 trafficking are considered critical in the development of neuropathic pain. SUMOylation of collapsin response mediator protein 2 (CRMP2) regulates the membrane trafficking and function of Na V 1.7. Enhanced CRMP2 SUMOylation in neuropathic pain correlates with increased Na V 1.7 activity. Pharmacological and genetic interventions that interfere with CRMP2 SUMOylation in rodents with neuropathic pain have been shown to reverse mechanical allodynia. Sentrin or SUMO-specific proteases (SENPs) are vital for balancing SUMOylation and deSUMOylation of substrates. Overexpression of SENP1 and/or SENP2 in CRMP2-expressing cells results in increased deSUMOylation and decreased membrane expression and currents of Na V 1.7. Although SENP1 is present in the spinal cord and dorsal root ganglia, its role in regulating Na V 1.7 function and pain is not known. We hypothesized that favoring SENP1 expression can enhance CRMP2 deSUMOylation to modulate Na V 1.7 channels. In this study, we used a clustered regularly interspaced short palindromic repeats activation (CRISPRa) SENP1 lentivirus to overexpress SENP1 in dorsal root ganglia neurons. We found that SENP1 lentivirus reduced CRMP2 SUMOylation, Na V 1.7-CRMP2 interaction, and Na V 1.7 membrane expression. SENP1 overexpression decreased Na V 1.7 currents through clathrin-mediated endocytosis, directly linked to CRMP2 deSUMOylation. Moreover, enhancing SENP1 expression did not affect the activity of TRPV1 channels or voltage-gated calcium and potassium channels. Intrathecal injection of CRISPRa SENP1 lentivirus reversed mechanical allodynia in male and female rats with spinal nerve injury. These results provide evidence that the pain-regulating effects of SENP1 overexpression involve, in part, the modulation of Na V 1.7 channels through the indirect mechanism of CRMP2 deSUMOylation.

SARS-CoV-2 Induces Epithelial-Enteric Neuronal Crosstalk Stimulating VIP Release.

Diarrhea is present in up to 30-50% of patients with COVID-19. The mechanism of SARS-CoV-2-induced diarrhea remains unclear. We hypothesized that enterocyte-enteric neuron interactions were important in SARS-CoV-2-induced diarrhea. SARS-CoV-2 induces endoplasmic reticulum (ER) stress in enterocytes causing the release of damage associated molecular patterns (DAMPs). The DAMPs then stimulate the release of enteric neurotransmitters that disrupt gut electrolyte homeostasis. Primary mouse enteric neurons (EN) were exposed to a conditioned medium from ACE2-expressing Caco-2 colonic epithelial cells infected with SARS-CoV-2 or treated with tunicamycin (ER stress inducer). Vasoactive intestinal peptides (VIP) expression and secretion by EN were assessed by RT-PCR and ELISA, respectively. Membrane expression of NHE3 was determined by surface biotinylation. SARS-CoV-2 infection led to increased expression of BiP/GRP78, a marker and key regulator for ER stress in Caco-2 cells. Infected cells secreted the DAMP protein, heat shock protein 70 (HSP70), into the culture media, as revealed by proteomic and Western analyses. The expression of VIP mRNA in EN was up-regulated after treatment with a conditioned medium of SARS-CoV-2-infected Caco-2 cells. CD91, a receptor for HSP70, is abundantly expressed in the cultured mouse EN. Tunicamycin, an inducer of ER stress, also induced the release of HSP70 and Xbp1s, mimicking SARS-CoV-2 infection. Co-treatment of Caco-2 with tunicamycin (apical) and VIP (basolateral) induced a synergistic decrease in membrane expression of Na+/H+ exchanger (NHE3), an important transporter that mediates intestinal Na+/fluid absorption. Our findings demonstrate that SARS-CoV-2 enterocyte infection leads to ER stress and the release of DAMPs that up-regulates the expression and release of VIP by EN. VIP in turn inhibits fluid absorption through the downregulation of brush-border membrane expression of NHE3 in enterocytes. These data highlight the role of epithelial-enteric neuronal crosstalk in COVID-19-related diarrhea.

Membrane pools of phosphatidylinositol-4-phosphate regulate KCNQ1/KCNE1 membrane expression

Plasma membrane phosphatidylinositol 4-phosphate (PI4P) is a precursor of PI(4,5)P2, an important regulator of a large number of ion channels. Although the role of the phospholipid PI(4,5)P2 in stabilizing ion channel function is well established, little is known about the role of phospholipids in channel membrane localization and specifically the role of PI4P in channel function and localization. The phosphatidylinositol 4-kinases (PI4Ks) synthesize PI4P. Our data show that inhibition of PI4K and prolonged decrease of levels of plasma membrane PI4P lead to a decrease in the KCNQ1/KCNE1 channel membrane localization and function. In addition, we show that mutations linked to Long QT syndrome that affect channel interactions with phospholipids lead to a decrease in membrane expression. We show that expression of a LQT1-associated C-terminal deletion mutant abolishes PI4Kinase-mediated decrease in membrane expression and rescues membrane expression for phospholipid-targeting mutations. Our results indicate a novel role for PI4P on ion channel regulation. Our data suggest that decreased membrane PI4P availability to the channel, either due to inhibition of PI4K or as consequence of mutations, dramatically inhibits KCNQ1/KCNE1 channel membrane localization and current. Our results may have implications to regulation of other PI4P binding channels.

Inhibition of Arginine Methylation Impairs Platelet Function.

Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups to arginine residues in proteins. PRMT inhibitors are novel, promising drugs against cancer that are currently in clinical trials, which include oral administration of the drugs. However, off-target activities of systemically available PRMT inhibitors have not yet been investigated. In this work, we study the relevance of arginine methylation in platelets and investigate the effect of PRMT inhibitors on platelet function and on the expression of relevant platelet receptors. We show that (1) key platelet proteins are modified by arginine methylation; (2) incubation of human platelets with PRMT inhibitors for 4 h results in impaired capacity of platelets to aggregate in response to thrombin and collagen, with IC50 values in the μM range; and (3) treatment with PRMT inhibitors leads to decreased membrane expression and reduced activation of the critical platelet integrin αIIbβ3. Our contribution opens new avenues for research on arginine methylation in platelets, including the repurposing of arginine methylation inhibitors as novel antiplatelet drugs. We also recommend that current and future clinical trials with PRMT inhibitors consider any adverse effects associated with platelet inhibition of these emerging anticancer drugs.

Abstract 2412: Decursin inhibits tumor progression in head and neck squamous cell cancer by down regulating CXCR7 expression

Abstract Background: CX chemokine receptor 7 (CXCR7) is frequently overexpressed in a variety of tumors and plays a significant role in tumor growth and metastasis. Thus, inhibition of CXCR7 is an important potential anticancer treatment strategy. Decursin, a derivative from Angelica gigas Nakai has been reported to have anti-tumor activity in various cancers. However, little is known about its anti-tumor activity and underlying mechanisms of action in head and neck squamous cell cancer (HNSCC). Methods: CXCR7-overexpressing HNSCC cell lines were established in vitro. The effects of decursin on cell growth, motility, migration, and invasion were investigated using CCK-8, wound healing, and transwell assays. In addition, the mechanism by which decusin shows antitumor activity was investigated. Results: Overexpression of CXCR7 significantly increased cell proliferation, migration, invasion, and cell motility compared with control cells. Treatment with decursin decreased membrane expression of CXCR7 in a concentration dependent manner and significantly inhibited tumor progression by suppressing cell growth, migration, invasion, and motility. Decursin was found to induce G0/G1 cell cycle arrest and regulate cell cycle-related proteins such as Cyclin A and CDK2 in HNSCC cells. In addition, decursin inhibited CXCR7-induced STAT3/c-MYC activation. Conclusion: These results demonstrate that CXCR7 promotes cancer progression through the STAT3/c-Myc pathway in HNSCC and that decursin is a natural product that may target CXCR7 for HNSCC treatment. Citation Format: Mina Joo, Solbi Kim, Nayoung Kim, Myung-Won Lee, Heung Jin Jeon, Hyewon Ryu, Ik-Chan Song, Jin-Man Kim, Hyo Jin Lee. Decursin inhibits tumor progression in head and neck squamous cell cancer by down regulating CXCR7 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2412.

The Intracellular N-Terminal Domain of the Acid-Sensing Ion Channel 1a Participates in Channel Opening and Membrane Expression.

Acid-sensing ion channels (ASICs) are widely expressed in the nervous system. The intracellular C terminus of ASIC1a has many sites involved in regulating its expression and the opening mechanism, but the role of the intracellular N-terminal domain is poorly understood. Here, we explored the correlation of ASIC1a intracellular N terminus with membrane expression and gate opening. We modified the N-terminal structure of ASICs by deletion/truncation/mutation strategies and transfected the recombinant plasmids into CHO cells. Protein expression was analyzed with immunofluorescence, Western blots, and patch-clamp experiments. Deleting the entire N terminus decreased the membrane expression of channel proteins, and ion channel opening was lost. Deleting sections of the N terminus also decreased membrane expression and suggested that all areas were significant, with no single or group of amino acid residues playing a decisive role in regulating ASIC1a membrane expression. In terms of gate opening, five amino acid (AA) residues from AA 16 to AA 20 participated in gate opening, and isoleucine at AA 18 was the most important. The whole N terminus of ASICs participates in the membrane expression of ASIC1a, and five amino acid residues (AA 16-20) are involved in the gate opening mechanism. SIGNIFICANCE STATEMENT: The whole N terminus of ASICs participates in the membrane expression of ASIC1a, and five amino acid resi-dues (amino acid 16-20) are involved in the gate opening mechanism.

Proteolytic activation of the epithelial sodium channel: role of pro‐protein convertases and prostasin

The epithelial sodium channel (ENaC) is essential for regulating renal sodium excretion. Cleavage of the γENaC subunit increases ENaC activity both in vitro and in vivo; however, the involved endogenous proteases remain ambiguous. This study investigates the mechanistic roles of the pro‐protein convertase (PCSK) family and the serine protease prostasin in proteolytic activation of ENaC.We used the M‐1 mouse collecting duct cell line that natively expresses functional ENaC channels for pharmacological inhibition studies with pro‐protein convertase inhibitor decanoyl‐RVKR‐chloromethyl ketone (dec‐RVKR‐cmk) and serine protease inhibitor camostat mesilate (CM). Knockout cell lines of prostasin and three PCSKs were generated with the CRISPR/Cas9 system in M‐1 cells. As an index of ENaC activity, amiloride‐sensitive short‐circuit current (ISC) and transepithelial voltage (VTE) was measured on polarized cells on transwell filter supports in Ussing chambers. γENaC membrane levels and cleavage were tested by western blotting of surface biotinylated M‐1 cells with an antibody against the C‐terminal of γENaC. All values are means SE, compared with students t‐test or ANOVA as appropriate.Pro‐protein convertase inhibition decreased the amiloride‐sensitive ISC in M‐1 cells compared with controls (25.9 1.9 μA, n=5, vs. 45.7 7.0 μA, n=6, p<0.05). Similarly, serine protease inhibition decreased VTE (−34.5 7.8 mV, n=3, vs. −74.2 0.9 mV, n=3, p<0.05) but ISC was not significantly lower (68.0 8.5, n=3, vs. 74.0 2.3, n=3, n.s.). Preliminary evidence suggests that although both inhibitors decreased membrane expression of γENaC, only serine protease inhibitor treatment reduced γENaC cleavage (n=1). Knockout of the Psck3 (furin) gene by CRISPR completely abolished ENaC activity, while knockout of Pcsk4, Pcsk7, and prostasin all showed decreased ENaC activity compared to wild‐type M‐1 cells. Knockout of PCSKs reduced γENaC surface expression, but only knockout of prostasin reduced γENaC cleavage.We propose that PCSKs are not directly involved in proteolytic ENaC activation. Both pharmacological inhibition and knockout of prostasin decreased ENaC activity and γENaC cleavage, thus suggesting a potential role in proteolytic ENaC activation.Support or Funding Information The Danish Medical Research Council

PKCβII specifically regulates KCNQ1/KCNE1 channel membrane localization

The slow voltage-gated potassium channel (IKs) is composed of the KCNQ1 and KCNE1 subunits and is one of the major repolarizing currents in the heart. Activation of protein kinase C (PKC) has been linked to cardiac arrhythmias. Although PKC has been shown to be a regulator of a number of cardiac channels, including IKs, little is known about regulation of the channel by specific isoforms of PKC. Here we studied the role of different PKC isoforms on IKs channel membrane localization and function. Our studies focused on PKC isoforms that translocate to the plasma membrane in response to Gq-coupled receptor (GqPCR) stimulation: PKCα, PKCβI, PKCβII and PKCε. Prolonged stimulation of GqPCRs has been shown to decrease IKs membrane expression, but the specific role of each PKC isoform is unclear. Here we show that stimulation of calcium-dependent isoforms of PKC (cPKC) but not PKCε mimic receptor activation. In addition, we show that general PKCβ (LY-333531) and PKCβII inhibitors but not PKCα or PKCβI inhibitors blocked the effect of cPKC on the KCNQ1/KCNE1 channel. PKCβ inhibitors also blocked GqPCR-mediated decrease in channel membrane expression in cardiomyocytes. Direct activation of PKCβII using constitutively active PKCβII construct mimicked agonist-induced decrease in membrane expression and channel function, while dominant negative PKCβII showed no effect. This suggests that the KCNQ1/KCNE1 channel was not regulated by basal levels of PKCβII activity. Our results indicate that PKCβII is a specific regulator of IKs membrane localization. PKCβII expression and activation are strongly increased in many disease states, including heart disease and diabetes. Thus, our results suggest that PKCβII inhibition may protect against acquired QT prolongation associated with heart disease.

Differential regulation of vacuolar H+‐ATPase subunits by Transforming Growth Factor beta 1 (TGF‐β1) in salivary ducts

Bicarbonate concentration in saliva is controlled by the action of acid‐base transporters in salivary duct cells. We have previously shown expression of ATP6V1E1 and ATP6V1A in human and rodent salivary ducts and adaptive redistribution to the apical plasma membrane following acute and chronic metabolic acidosis. However, whether such redistribution has any physiological significance has not been elucidated. Using the human submandibular gland cell line HSG, we show for the first time functional expression of V‐ATPase in submandibular duct cells and introduce Transforming Growth Factor beta (TGF‐β) as a novel regulator of V‐ATPase subunits. Using QRT‐PCR, immunoblotting, biotinylation of surface proteins, immunofluorescence, and intracellular H(+) recording with H(+) ‐sensitive dye 2′,7′‐bis‐(carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein, we show that activation of TGF‐β signalling upregulates ATP6V1E1 and ATP6V1B2, and has no effect on ATP6V1A. Following acidosis, neutralization of TGF‐βs or blocking of Smad2/3 phosphorylation decreased membrane expression of ATP6V1A, and prevented the acidosis‐induced increased V‐ATPase activity. The results suggest multiple modes of action of TGF‐β1 on V‐ATPase subunits in HSG cells: TGF‐β1 may regulate transcription or protein synthesis of certain subunits and trafficking of other subunits in a context‐dependent manner. Moreover, V‐ATPase is active in salivary duct cells and involved in intracellular pH regulation following acidosis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

3012 Functional consequences of pravastatin isomerization on OATP1B1-mediated transport

OBJECTIVES/SPECIFIC AIMS: In the present study, we examined the functional consequences of 3α-PVA on OATP1B1-mediated PVA transport. To elucidate this, we determined the effect of SLCO1B1 genotype on PVA transport and the role of 3αPVA as a competitive inhibitor of OATP1B1, which could serve as another covariate that disrupts the systemic and hepatic exposure of pravastatin in children and adults. METHODS/STUDY POPULATION: Site directed mutagenesis was performed to generate SLCO1B1 genotypes of interest (*1a, *1b, *5, *15). Human embryonic kidney (HEK293) cells were grown and plated at 200 000 cells per well in 24-well plates. Twenty-four hours later the cells were transfected with the aforementioned plasmids. Forty-eight hours later cell-based transport was performed with radiolabelled [3H]-pravastatin sodium salt. Non-radioactive pravastatin sodium salt and 3’α-iso-pravastatin sodium salt was used for PVA transport and 3αPVA studies, respectively. Cells were washed 3 times with warm uptake buffer, incubated for 1 minute with uptake solutions containing PVA and 3αPVA at varying concentrations. Transport was terminated by four 1-ml washes with ice-cold uptake buffer. Cells were lysed with 300 µl 1% Triton X-100 in PBS at room temperature for 30 minutes prior to analysis. Radioactivity was measured in a MicroBeta2 liquid scintillation counter. The remaining cell lysates were transferred to 96-well plates to determine total protein concentration using the bicinchonic acid protein assay. All transport measurements were corrected by the total protein concentration. All experiments were performed 3 to 4 times independently with 2-3 determinations. Data were analyzed for significant differences amongst genotype groups using ANOVA followed by Tukey’s multiple comparisons test. IC50 and kinetic parameters were calculated using non-linear regression analysis. RESULTS/ANTICIPATED RESULTS: Pravastatin transport in SLCO1B1 variants (*5, *15) was significantly decreased compared to the reference genotype *1a and *1b (Km [µM]: *1a 18.2 ± 0.9; *1b 17.9 ± 3.3; *5 34.2 ± 9.7; *15 34.1 ± 6.1; p≤0.05; Vmax [pmol/mg/min]: *1a 104.9 ± 13.1; *1b 93.7 ± 16.7; *5 44.8 ± 15.9; *15 62.3 ± 22.5; p≤0.05). *1a and *1b were not significantly different with respect to pravastatin transport. Intrinsic clearance was diminished nearly 4 to 5-fold in SLCO1B1 variants compared to reference genotypes (Vmax/Km [µl/min/mg]: *1a 5.8 ± 0.8; *1b 5.7 ± 1.9; *5 1.3 ± 0.2; *15 1.8 ± 0.3; p≤0.01). Pravastatin transport was inhibited by 3αPVA for all genotypes. However, there was more pronounced inhibition in the SLCO1B1 variant genotypes compared to reference genotypes (IC50 [µM]: *1a 15.9 ± 1.9; *1b 18.6 ± 5.7; *5 3.9 ± 2.0; *15 4.4 ± 0.8; p≤0.01; Ki: *1a 15.0 ± 1.8; *1b 17.5 ± 5.4; *5 3.8 ± 2.9; *15 4.3 ± 0.8; p≤0.01). DISCUSSION/SIGNIFICANCE OF IMPACT: In vitro PVA transport is altered according to SLCO1B1 genotype, consistent with previous in vitro and human experience. Our data suggest that the significantly different maximal transport velocity (Vmax) in variant versus non-variant genotypes is consistent with decreased membrane expression of OATP1B1 with the variant c.521T>C allele. However, in contrast to data involving typical model substrates (e.g. estrone-3-sulfate), the PVA binding affinity (Km) was significantly different between variant and non-variant genotypes, consistent with altered binding of the substrate to OATP1B1. Collectively, we conclude that decreased OATP1B1 expression and function in variant genotypes influence altered transport for PVA. Finally, the functional consequences of 3αPVA formation on PVA transport was confirmed in our study. Mechanistically, we confirmed our observation in humans that 3αPVA inhibits OATP1B1 transport. However, this effect is more pronounced in variant genotypes as shown by lower IC50 values compared to the reference genotypes. This highlights another source of variation that must be taken into consideration when trying to optimize the pravastatin dose-exposure relationship in humans.

Differential regulation of vacuolar H+ -ATPase subunits by transforming growth factor-β1 in salivary ducts.

Bicarbonate concentration in saliva is controlled by the action of acid-base transporters in salivary duct cells. We show for the first time expression of ATP6V1B1 in submandibular gland and introduce transforming growth factor-beta (TGF-β) as a novel regulator of V-ATPase subunits. Using QRT-PCR, immunoblotting, biotinylation of surface proteins, immunofluorescence, chromatin immunoprecipitation, and intracellular H(+ ) recording with H(+ )-sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein we show that in the human submandibular gland (HSG) cell line, activation of TGF-β signaling upregulates ATP6V1E1 and ATP6V1B2, downregulates ATP6V1B1, and has no effect on ATP6V1A. TGF-β1 effects on ATP6V1B1 are mediated through the canonical, the soluble adenylate cyclase, and ERK signaling. A CREB binding sequence was identified in the ATP6V1B1 promoter and CREB binding decreased after TGF-β1 treatment. Following acidosis, a bafilomycin-sensitive and Na+ -independent cell pH recovery was observed in HSG cells, an effect that was not influenced after disruption of acidic lysosomes. Moreover, neutralization of TGF-βs, inhibition of TGF-β receptor, or inhibition of the canonical pathway decreased membrane expression of ATP6V1A and prevented the acidosis-induced increased V-ATPase activity. The results suggest multiple modes of action of TGF-β1 on V-ATPase subunits in HSG cells: TGF-β1 may regulate transcription or protein synthesis of certain subunits and trafficking of other subunits in a context-dependent manner. Moreover, surface V-ATPase is active in salivary duct cells and involved in intracellular pH regulation following acidosis.

Structural mechanisms for defective CFTR gating caused by the Q1412X mutation, a severe ClassVI pathogenic mutation in cystic fibrosis.

Electrophysiological characterization of Q1412X-CFTR, a C-terminal truncation mutation of cystic fibrosis transmembrane conductance regulator (CFTR) associated with the severe form of cystic fibrosis (CF), reveals a gating defect that has not been reported previously. Mechanistic investigations of the gating deficit in Q1412X-CFTR suggest that the reduced open probability in Q1412X-CFTR is the result of a disruption of the function of the second ATP binding site (or site 2) in the nucleotide binding domains (NBDs). Detailed comparisons of several mutations with different degrees of truncation in the C-terminal region of NBD2 reveal the importance of the last two beta-strands in NBD2 for maintaining proper gating functions. The results of the present study also show that the application of clinically-approved drugs (VX-770 and VX-809) can greatly enhance the function of Q1412X, providing invitro evidence for a therapeutic strategy employing both reagents for patients bearing Q1412X or similar truncation mutations. Cystic fibrosis (CF) is caused by loss-of-function mutations of cystic fibrosis transmembrane conductance regulator (CFTR), a phosphorylation-activated but ATP-gated chloride channel. Based on the molecular mechanism of CF pathogenesis, disease-associated mutations are categorized into six classes. Among them, ClassVI, whose members include some of the C-terminal truncation mutations such as Q1412X, is defined as decreased membrane expression because of a faster turnover rate. In the present study, we characterized the functional properties of Q1412X-CFTR, a severe-form premature stop codon mutation. We confirmed previous findings of a ∼90% decrease in membrane expression but found a ∼95% reduction in the open probability (Po ). Detailed kinetic studies support the idea that the gating defect is the result of a dysfunctional ATP-binding site 2 in the nucleotide binding domains (NBDs). Because the Q1412X mutation results in a deletion of the last two beta-strands in NBD2 and the whole C-terminal region, we further characterized truncation mutations with different degrees of deletion in this segment. Mutations that completely or partially remove the C-terminus of CFTR at the same time as keeping an intact NBD2 (i.e. D1425X and S1455X) assume gating function almost identical to that of wild-type channels. However, the deletion of the last beta-strand in the NBD2 (i.e. N1419X) causes gating dysfunction that is milder than that of Q1412X. Thus, normal CFTR gating requires structural integrity of NBD2. Moreover, our observation that clinically-approved VX-809 (Lumacaftor, Vertex Pharmaceuticals, Boston, MA, USA) and VX-770 (Ivacaftor, Vertex Pharmaceuticals, Boston, MA, USA) significantly enhance the overall function of Q1412X-CFTR provides the conceptual basis for the treatment of patients carrying this mutation.

Abstract 4176: Abrogation of desmoglein 2-mediated c-met activation as a novel therapeutic strategy for reducing anaplastic thyroid cancer metastasis

Abstract Anaplastic thyroid cancer (ATC) is a fast growing and highly metastatic cancer with limited therapeutic options. Thus, targeted therapies need to be developed. This study aims to determine desmoglein 2 (Dsg2) expression in ATC and to elucidate its biological role and potential as a therapeutic target in ATC. Dsg2 expression was low or abnormal in ATC tissues. The ATC patients with reduced Dsg2 expression also had distant metastasis. Dsg2 depletion significantly increased cell migration and invasion, with relatively little effect on ATC cell proliferation in vitro, and increased distant metastasis in vivo. Dsg2 knockdown induced cell motility through the hepatocyte growth factor receptor (HGFR, c-Met)/Src/Rac1 signaling axis, with no alterations in the expression of EMT-related molecules. Further, specific targeting of c-Met significantly inhibited the motility of shDsg2-depleted ATC cells. Decreased membrane expression of Dsg2 increases the metastatic potential of ATC cells. Dsg2 plays an important role in migration and invasiveness in cell lines derived from ATC. Therapies targeting c-Met might be effective for ATC patients with decreased membrane expression of Dsg2. Thus, analysis of Dsg2 expression may provide a novel therapeutic strategy for ATC treatment. Keywords Anaplastic Thyroid Cancer, Desmoglein 2, Metastasis, c-Met, Tivantinib Citation Format: Dong G. Lee, Jeong-Ki Min. Abrogation of desmoglein 2-mediated c-met activation as a novel therapeutic strategy for reducing anaplastic thyroid cancer metastasis [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 4176.

Cadherin Expression Shift Could Well Distinguish Esophageal Squamous Cell Carcinoma from Non- Cancerous Esophageal Tissues

Background: Biomarkers for esophageal squamous cell carcinoma (ESCC) identification with high sensitivity are not well established. Since abnormal expression of cadherins has been widely reported in cancer, we explored its feasibility as an ESCC biomarker. Methods: Expression of E-cadherin and N-cadherin were detected in 150 esophageal tissues by immunohistochemistry. Staining strength and percentage in different subcellular structures of each specimen were evaluated by 2 independent pathologists. A logistic regression-based classifier derived from E-cadherin and N-cadherin staining was generated. Results: E-cadherin exhibited decreased membrane expression in ESCC, while N-cadherin exhibited decreased expression in the nucleus but elevated expression in the cytoplasm. Both E-cadherin and N-cadherin could distinguish ESCC tissues from non-cancerous tissues (area under the curve (AUC) = 0.748, 0.801, respectively). E-cadherin and N-cadherin staining scores could be merged into a cadherin (CDH) logistic index, which showed better discrimination (AUC = 0.909) than E-cadherin or N-cadherin alone. Further investigation indicated that the CDH logistic index was significantly correlated with tumor size and differentiation in ESCC. Conclusion: Both E-cadherin and N-cadherin had a strong expression shift in ESCC compared with non-cancerous tissues. The CDH logistic index, a parameter integrating the expression data of both cadherins, could be used as a marker with high sensitivity and specificity in the identification of ESCC.

RNAi-mediated human Nestin silence inhibits proliferation and migration of malignant melanoma cells by G1/S arrest via Akt-GSK3β-Rb pathway.

Human Nestin (hNestin) has been found to express in melanoma, and its expression is positively correlated with the advanced stage of melanoma. However, the precise role of hNestin in the development of melanoma has not been fully understood. The present study aimed to explore the role of hNestin in the proliferation and invasion of melanoma cells. The lentivirus vector carrying a short hairpin RNAs (shRNAs) targeting hNestin (hNestin-shRNA-LV) was stably infected into human melanoma cells UACC903, which expressed high levels of hNestin. The effects of hNestin knockdown on the proliferation, apoptosis, migration of melanoma cells and the related signaling pathways were investigated by immunofluorence, Western blotting and reverse transcription polymerase chain reaction (RT-PCR), respectively. The results showed that hNestin was expressed in most melanoma specimens and the melanoma cells studied. Knockdown of hNestin expression significantly inhibited the proliferation of melanoma cells, blocked the formation of cell colony, arrested cell cycle at G1/S stage and suppressed the activation of Akt and GSK3β. hNestin-silent cells also showed a sheet-like appearance with tight cell-cell adhesion, decreased membrane expression of N-cadherin and β-catenin, and attenuated migration. Furthermore, hNestin silence resulted in the inhibition of tumor growth in vivo. Our study indicates that hNestin knockdown suppresses the proliferation of melanoma cells, which might be through affecting Akt-GSK3β-Rb pathway-mediated G1/S arrest, and hNestin silence inhibits the migration by selectively modulating the expression of cell adhesion molecules in the process of epithelial-mesenchymal transition.

Benzo(a)pyrene triggers desensitization of \u03b22-adrenergic pathway

Exposure to environmental polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (B(a)P), has been linked to several health-threatening risks. PAHs were also shown to hinder adrenergic receptor (ADR) responses. As we previously demonstrated that B(a)P can directly interact with the β2ADR, we investigated here whether B(a)P could decrease β2ADR responsiveness by triggering receptor desensitization phenomena. We firstly showed that exposure to B(a)P reduced β2ADR-mediated epinephrine-induced induction of NR4A gene mRNAs and of intracellular cAMP. Analysis of β2ADR protein expression demonstrated that B(a)P rapidly decreased membrane expression of β2ADR with a subsequent degradation of receptor protein. B(a)P exposure concomitantly rapidly increased the β2ADR mRNA levels. The use of the β-blockers, propranolol and ICI 118.551, demonstrated the involvement of β2ADR itself in this increase. However, sustained exposure to B(a)P induced a diminution of β2ADR mRNA steady-state as a result of the acceleration of its degradation. Together, these results show that, beside the well-known activation of the aryl hydrocarbon receptor, PAH deleterious effects may involve the dysfunction of adrenergic responses through, in part, the desensitization of β2ADR. This may be taken in consideration when β2-agonists/antagonists are administered in patients exposed to important concentrations of PAHs, e.g. in cigarette smokers.

Small interference ITGA6 gene targeting in the human thymic epithelium differentially regulates the expression of immunological synapse-related genes

ABSTRACTThe thymus supports differentiation of T cell precursors. This process requires relocation of developing thymocytes throughout multiple microenvironments of the organ, mainly with thymic epithelial cells (TEC), which control intrathymic T cell differentiation influencing the formation and maintenance of the immunological synapse. In addition to the proteins of the major histocompatibility complex (MHC), this structure is supported by several adhesion molecules. During the process of thymopoiesis, we previously showed that laminin-mediated interactions are involved in the entrance of T-cell precursors into the thymus, as well as migration of differentiating thymocytes within the organ. Using small interference RNA strategy, we knocked-down the ITGA6 gene (which encodes the CD49f integrin α-chain) in cultured human TEC, generating a decrease in the expression of the corresponding CD49f subunit, in addition to modulation in several other genes related to cell adhesion and migration. Thymocyte adhesion to TEC was significantly impaired, comprising both immature and mature thymocyte subsets. Moreover, we found a modulation of the MHC, with a decrease in membrane expression of HLA-ABC, in contrast with increase in the expression of HLA-DR. Interestingly, the knockdown of the B2M gene (encoding the β-2 microglobulin of the HLA-ABC complex) increased CD49f expression levels, thus unraveling the existence of a cross-talk event in the reciprocal control of CD49f and HLA-ABC. Our data suggest that the expression levels of CD49f may be relevant in the general control of MHC expression by TEC and consequently the corresponding synapse with developing thymocytes mediated by the T-cell receptor.

Arrhythmia causes lipid accumulation and reduced glucose uptake.

Atrial fibrillation (AF) is characterized by irregular contractions of atrial cardiomyocytes and increased energy demand. The aim of this study was to characterize the influence of arrhythmia on glucose and fatty acid (FA) metabolism in cardiomyocytes, mice and human left atrial myocardium. Compared to regular pacing, irregular (pseudo-random variation at the same number of contractions/min) pacing of neonatal rat cardiomyocytes induced shorter action potential durations and effective refractory periods and increased diastolic [Ca(2+)]c. This was associated with the activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and AMP-activated protein kinase (AMPK). Membrane expression of fatty acid translocase (FAT/CD36) and (14)C-palmitic acid uptake were augmented while membrane expression of glucose transporter subtype 4 (GLUT-4) as well as (3)H-glucose uptake were reduced. Inhibition of AMPK and CaMKII prevented these arrhythmia-induced metabolic changes. Similar alterations of FA metabolism were observed in a transgenic mouse model (RacET) for spontaneous AF. Consistent with these findings samples of left atrial myocardium of patients with AF compared to matched samples of patients with sinus rhythm showed up-regulation of CaMKII and AMPK and increased membrane expression of FAT/CD36, resulting in lipid accumulation. These changes of FA metabolism were accompanied by decreased membrane expression of GLUT-4, increased glycogen content and increased expression of the pro-apoptotic protein bax. Irregular pacing of cardiomyocytes increases diastolic [Ca(2+)]c and activation of CaMKII and AMPK resulting in lipid accumulation, reduced glucose uptake and increased glycogen synthesis. These metabolic changes are accompanied by an activation of pro-apoptotic signalling pathways.