Diphyllin Shows a Broad-Spectrum Antiviral Activity against Multiple Medically Important Enveloped RNA and DNA Viruses.

Diphyllin is a natural arylnaphtalide lignan extracted from tropical plants of particular importance in traditional Chinese medicine. This compound has been described as a potent inhibitor of vacuolar (H+)ATPases and hence of the endosomal acidification process that is required by numerous enveloped viruses to trigger their respective viral infection cascades after entering host cells by receptor-mediated endocytosis. Accordingly, we report here a revised, updated, and improved synthesis of diphyllin, and demonstrate its antiviral activities against a panel of enveloped viruses from Flaviviridae, Phenuiviridae, Rhabdoviridae, and Herpesviridae families. Diphyllin is not cytotoxic for Vero and BHK-21 cells up to 100 µM and exerts a sub-micromolar or low-micromolar antiviral activity against tick-borne encephalitis virus, West Nile virus, Zika virus, Rift Valley fever virus, rabies virus, and herpes-simplex virus type 1. Our study shows that diphyllin is a broad-spectrum host cell-targeting antiviral agent that blocks the replication of multiple phylogenetically unrelated enveloped RNA and DNA viruses. In support of this, we also demonstrate that diphyllin is more than just a vacuolar (H+)ATPase inhibitor but may employ other antiviral mechanisms of action to inhibit the replication cycles of those viruses that do not enter host cells by endocytosis followed by low pH-dependent membrane fusion.

Melatonin Attenuates RANKL-Induced Osteoclastogenesis via Inhibition of Atp6v0d2 and DC-STAMP through MAPK and NFATc1 Signaling Pathways.

Melatonin is a hormone secreted by the pineal gland that is involved in the biorhythm of reproductive activities. The present study investigated the inhibitory effects of melatonin on osteoclastogenesis in RAW 264.7 cells according to changes in V-ATPase and the corresponding inhibition of the MAPK and NFATc1 signaling processes. METHODS: the cytotoxic effect of melatonin was investigated by MTT assay. Osteoclast differentiation and gene expression of osteoclast-related factors were confirmed via TRAP staining, pit formation assay, immunofluorescence imaging, western blot, and real-time PCR. RESULTS: melatonin was found to inactivate the p38 and JNK of MAP kinase in RAW264.7 cells treated with RANKL and treated with a combination RANKL and melatonin for 1, 3, and 5 days. The melatonin treatment group showed a reduction in osteoclastogenesis transcription factors and ATP6v0d2 gene expression. CONCLUSIONS: melatonin inhibits osteoclast differentiation and cell fusion by inhibiting the expression of Atp6v0d2 through the inactivation of MAPK and NFATc1 signaling in RANKL-stimulated RAW264.7 macrophages. The findings of the present study suggest that melatonin could be a suitable therapy for bone loss and imply a potential role of melatonin in bone health.

miR-4454 Promotes Hepatic Carcinoma Progression by Targeting Vps4A and Rab27A.

Hepatocellular carcinoma (HCC) has high morbidity and mortality. MicroRNAs (miRNAs), which could be regulated by cancer-derived exosomes, play critical regulatory roles in the initiation and development of cancer. However, the expressions, effects, and mechanisms of abundant miRNAs regulated by HCC cancer-derived exosomes in HCC remain largely unclear. Exosomes of HepG2 cells under heat shock, TGF-ß1, doxorubicin, acid and hypoxia/reoxygenation (H/R) conditions, and exosomes were successfully identified by transmission electron microscopy and Western blot analysis. The identified exosomes were then applied to evaluate the miRNA expression profiles by RNA sequencing. Mechanically, we discovered that doxorubicin was upregulated, TGF-ß1 downregulated the expressions of Vps4A, Rab27A, Alix, and Hrs in HepG2 cells and exosomes, and Vps4A and Rab27A, as target genes for miR-4454, could also be downregulated by miR-4454. Functionally, we revealed that miR-4454 inhibitor and miR-4454 inhibitor-mediated exosomes could markedly suppress proliferation, migration, invasion, and vascularization and accelerate cycle arrest, apoptosis, and ROS of HepG2 cells. This study provided many potential HCC cancer-derived exosome-mediated miRNAs in HCC under 5 different stimulus conditions. Meanwhile, we certified that miR-4454 in exosomes could provide a novel and effective mechanism for HCC function.

Inhibiting ATP6V0D2 Aggravates Liver Ischemia-Reperfusion Injury by Promoting NLRP3 Activation via Impairing Autophagic Flux Independent of Notch1/Hes1.

At present, liver ischemia-reperfusion (IR) injury is still a great challenge for clinical liver partial resection and liver transplantation. The innate immunity regulated by liver macrophages orchestrates the cascade of IR inflammation and acts as a bridge. As a specific macrophage subunit of vacuolar ATPase, ATP6V0D2 (V-ATPase D2 subunit) has been shown to promote the formation of autophagolysosome in vitro. Our research fills a gap which has existed in the study of inflammatory stress about the V-ATPase subunit ATP6V0D2 in liver macrophages. We first found that the expression of specific ATP6V0D2 in liver macrophages was upregulated with the induction of inflammatory cascade after liver IR surgery, and knockdown of ATP6V0D2 resulted in increased secretion of proinflammatory factors and chemokines, which enhanced activation of NLRP3 and aggravation of liver injury. Further studies found that the exacerbated activation of NLRP3 was related to the autophagic flux regulated by ATP6V0D2. Knocking down ATP6V0D2 impaired the formation of autophagolysosome and aggravated liver IR injury through nonspecific V-ATPase activation independent of V-ATPase-Notchl-Hesl signal axis. In general, we illustrated that the expression of ATP6V0D2 in liver macrophages was upregulated after liver IR, and by gradually promoting the formation of autophagolysosomes to increase autophagy flux to limit the activation of liver inflammation, this regulation is independent of the Notch1-Hes1 signal axis.

Molecular basis of V-ATPase inhibition by bafilomycin A1.

Pharmacological inhibition of vacuolar-type H+-ATPase (V-ATPase) by its specific inhibitor can abrogate tumor metastasis, prevent autophagy, and reduce cellular signaling responses. Bafilomycin A1, a member of macrolide antibiotics and an autophagy inhibitor, serves as a specific and potent V-ATPases inhibitor. Although there are many V-ATPase structures reported, the molecular basis of specific inhibitors on V-ATPase remains unknown. Here, we report the cryo-EM structure of bafilomycin A1 bound intact bovine V-ATPase at an overall resolution of 3.6-Å. The structure reveals six bafilomycin A1 molecules bound to the c-ring. One bafilomycin A1 molecule engages with two c subunits and disrupts the interactions between the c-ring and subunit a, thereby preventing proton translocation. Structural and sequence analyses demonstrate that the bafilomycin A1-binding residues are conserved in yeast and mammalian species and the 7'-hydroxyl group of bafilomycin A1 acts as a unique feature recognized by subunit c.

V-Type ATPase Mediates Airway Surface Liquid Acidification in Pig Small Airway Epithelial Cells.

In a newborn pig cystic fibrosis (CF) model, the ability of gland-containing airways to fight infection was affected by at least two major host-defense defects: impaired mucociliary transport and a lower airway surface liquid (ASL) pH. In the gland-containing airways, the ASL pH is balanced by CFTR (CF transmembrane conductance regulator) and ATP12A, which, respectively, control HCO3- transport and proton secretion. We found that, although porcine small airway tissue expressed lower amounts of ATP12A, the ASL of epithelial cultures from CF distal small airways (diameter < 200 µm) were nevertheless more acidic (compared with non-CF airways). Therefore, we hypothesized that gland-containing airways and small airways control acidification using distinct mechanisms. Our microarray data suggested that small airway epithelia mediate proton secretion via ATP6V0D2, an isoform of the V0 d subunit of the H+-translocating plasma membrane V-type ATPase. Immunofluorescence of small airways verified the expression of the V0 d2 subunit isoform at the apical surface of Muc5B+ secretory cells, but not ciliated cells. Inhibiting the V-type ATPase with bafilomycin A1 elevated the ASL pH of small airway cultures, in the presence or absence of HCO3-, and decreased ASL viscosity. These data suggest that, unlike large airways, which are acidified by ATP12A activity, small airways are acidified by V-type ATPase, thus identifying V-type ATPase as a novel therapeutic target for small airway diseases.

V-ATPase Inhibition Decreases Mutant Androgen Receptor Activity in Castrate-resistant Prostate Cancer.

Prostate cancer is critically dependent on androgen receptor (AR) signaling. Despite initial responsiveness to androgen deprivation, most patients with advanced prostate cancer subsequently progress to a clinically aggressive castrate-resistant prostate cancer (CRPC) phenotype, typically associated with expression of splice-variant or mutant AR forms. Although current evidence suggests that the vacuolar-ATPase (V-ATPase), a multiprotein complex that catalyzes proton transport across intracellular and plasma membranes, influences wild-type AR function, the effect of V-ATPase inhibition on variant AR function is unknown.Inhibition of V-ATPase reduced AR function in wild-type and mutant AR luciferase reporter models. In hormone-sensitive prostate cancer cell lines (LNCaP, DuCaP) and mutant AR CRPC cell lines (22Rv1, LNCaP-F877L/T878A), V-ATPase inhibition using bafilomycin-A1 and concanamycin-A reduced AR expression, and expression of AR target genes, at mRNA and protein levels. Furthermore, combining chemical V-ATPase inhibition with the AR antagonist enzalutamide resulted in a greater reduction in AR downstream target expression than enzalutamide alone in LNCaP cells. To investigate the role of individual subunit isoforms, siRNA and CRISPR-Cas9 were used to target the V1C1 subunit in 22Rv1 cells. Whereas transfection with ATP6V1C1-targeted siRNA significantly reduced AR protein levels and function, CRISPR-Cas9-mediated V1C1 knockout showed no substantial change in AR expression, but a compensatory increase in protein levels of the alternate V1C2 isoform.Overall, these results indicate that V-ATPase dysregulation is directly linked to both hormone-responsive prostate cancer and CRPC via impact on AR function. In particular, V-ATPase inhibition can reduce AR signaling regardless of mutant AR expression.

Inhibition of lysosomal vacuolar proton pump down-regulates cellular acidification and enhances E. coli bactofection efficiency.

Endosomal escape is considered a crucial barrier that needs to be overcome by integrin-mediated E. coli for gene delivery into mammalian cells. Bafilomycin, a potent inhibitor of the H+ proton pump commonly employed to lower endosomal pH, was evaluated as part of the E. coli protocol during delivery. We found an increase in green fluorescent protein expression up 6.9, 3.2, 5.0, 2.8, and 4.5 fold in HeLa, HEK-293, A549, HT1080, and MCF-7 respectively, compared to untreated cells. Our result showed for the first time that Inhibition of lysosomal V-ATPase enhances E. coli efficiency.

Silencing of Central (Pro)renin Receptor Ameliorates Salt-Induced Renal Injury in Chronic Kidney Disease.

Aims: A high-salt diet can aggravate oxidative stress, and renal fibrosis via the brain and renal renin-angiotensin system (RAS) axis in chronic kidney disease (CKD) rats. (Pro)renin receptor (PRR) plays a role in regulating RAS and oxidative stress locally. However, whether central PRR regulates salt-induced renal injury in CKD remains undefined. Here, we hypothesized that the reduction of central PRR expression could ameliorate central lesions and thereby ameliorate renal injury in high-salt-load CKD rats. Results: We investigated RAS, sympathetic nerve activity, oxidative stress, inflammation, and tissue injury in subfornical organs and kidneys in high-salt-load 5/6 nephrectomy CKD rats after the silencing of central PRR expression by intracerebroventricular lentivirus-RNAi. We found that the sympathetic nerve activity was reduced, and the levels of inflammation and oxidative stress were decreased in both brain and kidney. Renal injury and fibrosis were ameliorated. To explore the mechanism by which central inhibition of PRR expression ameliorates kidney damage, we blocked central MAPK/ERK1/2 and PI3K/Akt signaling pathways as well as angiotensin converting enzyme 1-angiotensin II-angiotensin type 1 receptors (ACE1-Ang II-AT1R) axis. Salt-induced overexpression of renal RAS, inflammation, oxidative stress, and fibrosis in CKD rats were prevented by central blockade of the pathways. Innovation: This study provides new insights into the mechanisms underlying salt-induced kidney damage. Targeting central PRR or PRR-mediated signaling pathway may be a novel strategy for the treatment of CKD. Conclusions: These results suggested that the silencing of central PRR expression ameliorates salt-induced renal injury in CKD through Ang II-dependent and -independent pathways.

Callyspongiolide Is a Potent Inhibitor of the Vacuolar ATPase.

Callyspongiolide is a marine-derived macrolide that kills cells in a caspase-independent manner. NCI COMPARE analysis of human tumor cell line toxicity data for synthetic callyspongiolide indicated that its pattern of cytotoxicity correlated with that seen for concanamycin A, an inhibitor of the vacuolar-type H+-ATPase (V-ATPase). Using yeast as a model system, we report that treatment with synthetic callyspongiolide phenocopied a loss of V-ATPase activity including (1) inability to grow on a nonfermentable carbon source, (2) rescue of cell growth via supplementation with Fe2+, (3) pH-sensitive growth, and (4) a vacuolar acidification defect visualized using the fluorescent dye quinacrine. Crucially, in an in vitro assay, callyspongiolide was found to dose-dependently inhibit yeast V-ATPase (IC50 = 10 nM). Together, these data identify callyspongiolide as a new and highly potent V-ATPase inhibitor. Notably, callyspongiolide is the first V-ATPase inhibitor known to be expelled by Pdr5p.

In vivo anti-V-ATPase antibody treatment delays ovarian tumor growth by increasing antitumor immune responses.

Tumor acidity is the key metabolic feature promoting cancer progression and is modulated by pH regulators on a cancer cell's surface that pump out excess protons/lactic acid for cancer cell survival. Neutralizing tumor acidity improves the therapeutic efficacy of current treatments including immunotherapies. Vacuolar-ATPase (V-ATPase) proton pumps encompass unique plasma membrane-associated subunit isoforms, making this molecule an important target for anticancer therapy. Here, we examined the in vivo therapeutic efficacy of an antibody (a2v-mAB) targeting specific V-ATPase-'V0a2' surface isoform in controlling ovarian tumor growth. In vitro a2v-mAb treatment inhibited the proton pump activity in ovarian cancer (OVCA) cells. In vivo intraperitoneal a2v-mAb treatment drastically delayed ovarian tumor growth with no measurable in vivo toxicity in a transplant tumor model. To explore the possible mechanism causing delayed tumor growth, histochemical analysis of the a2v-mAb-treated tumor tissues displayed high immune cell infiltration (M1-macrophages, neutrophils, CD103+ cells, and NK cells) and an enhanced antitumor response (iNOS, IFN-y, IL-1α) compared to control. There was marked decrease in CA-125-positive cancer cells and an enhanced active caspase-3 expression in a2v-mAb-treated tumors. RNA-seq analysis of a2v-mAb tumor tissues further revealed upregulation of apoptosis-related and toll-like receptor pathway-related genes. Indirect coculture of a2v-mAb-treated OVCA cells with human PBMCs in an unbuffered medium led to an enhanced gene expression of antitumor molecules IFN-y, IL-17, and IL-12-A in PBMCs, further validating the in vivo antitumor responses. In conclusion, V-ATPase inhibition using a monoclonal antibody directed against the V0a2 isoform increases antitumor immune responses and could therefore constitute an effective treatment strategy in OVCA.

Antiproliferative Effects of Monoclonal Antibodies against (Pro)Renin Receptor in Pancreatic Ductal Adenocarcinoma.

We previously reported that silencing of the PRR gene, which encodes the (pro)renin receptor [(P)RR], significantly reduced Wnt/ß-catenin-dependent development of pancreatic ductal adenocarcinoma (PDAC). Here, we examined the effects of a panel of blocking mAbs directed against the (P)RR extracellular domain on proliferation of the human PDAC cell lines PK-1 and PANC-1 in vitro and in vivo We observed that four rat anti-(P)RR mAbs induced accumulation of cells in the G0-G1-phase of the cell cycle and significantly reduced proliferation in vitro concomitant with an attenuation of Wnt/ß-catenin signaling. Systemic administration of the anti-(P)RR mAbs to nude mice bearing subcutaneous PK-1 xenografts significantly decreased tumor expression of active ß-catenin and the proliferation marker Ki-67, and reduced tumor growth. In contrast, treatment with the handle region peptide of (pro)renin did not inhibit tumor growth in vitro or in vivo, indicating that the effects of the anti-(P)RR mAbs were independent of the renin-angiotensin system. These data indicate that mAbs against human (P)RR can suppress PDAC cell proliferation by hindering activation of the Wnt/ß-catenin signaling pathway. Thus, mAb-mediated (P)RR blockade could be an attractive therapeutic strategy for PDAC.

Pharmacological Targeting of Vacuolar H+-ATPase via Subunit V1G Combats Multidrug-Resistant Cancer.

Multidrug resistance (MDR) in cancer remains a major challenge for the success of chemotherapy. Natural products have been a rich source for the discovery of drugs against MDR cancers. Here, we applied high-throughput cytotoxicity screening of an in-house natural product library against MDR SGC7901/VCR cells and identified that the cyclodepsipeptide verucopeptin demonstrated notable antitumor potency. Cytological profiling combined with click chemistry-based proteomics revealed that ATP6V1G directly interacted with verucopeptin. ATP6V1G, a subunit of the vacuolar H+-ATPase (v-ATPase) that has not been previously targeted, was essential for SGC7901/VCR cell growth. Verucopeptin exhibited strong inhibition of both v-ATPase activity and mTORC1 signaling, leading to substantial pharmacological efficacy against SGC7901/VCR cell proliferation and tumor growth in vivo. Our results demonstrate that targeting v-ATPase via its V1G subunit constitutes a unique approach for modulating v-ATPase and mTORC1 signaling with great potential for the development of therapeutics against MDR cancers.

Drug Sequestration in Lysosomes as One of the Mechanisms of Chemoresistance of Cancer Cells and the Possibilities of Its Inhibition.

Resistance to chemotherapeutics and targeted drugs is one of the main problems in successful cancer therapy. Various mechanisms have been identified to contribute to drug resistance. One of those mechanisms is lysosome-mediated drug resistance. Lysosomes have been shown to trap certain hydrophobic weak base chemotherapeutics, as well as some tyrosine kinase inhibitors, thereby being sequestered away from their intracellular target site. Lysosomal sequestration is in most cases followed by the release of their content from the cell by exocytosis. Lysosomal accumulation of anticancer drugs is caused mainly by ion-trapping, but active transport of certain drugs into lysosomes was also described. Lysosomal low pH, which is necessary for ion-trapping is achieved by the activity of the V-ATPase. This sequestration can be successfully inhibited by lysosomotropic agents and V-ATPase inhibitors in experimental conditions. Clinical trials have been performed only with lysosomotropic drug chloroquine and their results were less successful. The aim of this review is to give an overview of lysosomal sequestration and expression of acidifying enzymes as yet not well known mechanism of cancer cell chemoresistance and about possibilities how to overcome this form of resistance.

Can image analysis provide evidence that lysosomal sequestration mediates daunorubicin resistance?

Altered intracellular distribution of weak base anticancer drugs owing to lysosomal sequestration is one purported mechanism contributing to chemotherapy resistance. This has often been demonstrated with the example of daunorubicin (DNR), chemotherapy with its characteristic red fluorescence used to trace it in cellular compartments. Here we addressed the question whether image analysis of DNR fluorescence can reflect its real intracellular distribution. We observed that the relationship between the intensity of the DNR fluorescence and its concentration in water solutions with or without proteins is far from linear. In contrast, nucleic acids, RNA and DNA in particular, dramatically diminish the DNR fluorescence, however, the intensity was proportional to the amount. Therefore, image analysis reflects the composition of different cell compartments (i.e., the presence of proteins and nucleic acids) rather than the actual concentration of DNR in these compartments. In line with these results, we observed highly fluorescent lysosomes and low fluorescent nucleus in sensitive cancer cells treated with low DNR concentrations, a fluorescence pattern thought to be found only in resistant cancer cells. Importantly, LC/MS/MS analysis of extracts from sensitive cells treated with DNR or DNR in combination with an inhibitor of vacuolar ATPase, concanamycin A, indicated that lysosomal accumulation of DNR increased with increasing extracellular concentration. However, even the highest lysosomal accumulation of DNR failed to reduce its extralysosomal concentration and thus change the cell sensitivity to the drug. In conclusion, our results strongly suggest that DNR fluorescence within cells does not indicate the real drug distribution. Further they suggested that lysosomal sequestration of DNR can hardly contribute to its resistance in cancer cells in vitro.

V-ATPase blockade reduces renal gluconeogenesis and improves insulin secretion in type 2 diabetic rats.

Vacuolar H+-adenosine triphosphatase (V-ATPase) stimulates vesicular acidification that may activate cytoplasmic enzymes, hormone secretion and membrane recycling of transporters. We investigated the effect of blockade of V-ATPase by bafilomycin B1 on renal gluconeogenesis, mitochondrial enzymes, and insulin secretion in type 2 diabetic rats. Spontaneous type 2 diabetic Torii rats were treated with intraperitoneal injection of bafilomycin B1 for 1 week, and the kidneys were examined after 24 h of starvation in metabolic cages. The renal expression and activity of V-ATPase were increased in the brush border membrane of the proximal tubules in diabetic rats. The blockade of V-ATPase by bafilomycin B1 reduced renal V-ATPase activity and urinary ammonium in diabetic rats. Treatment with bafilomycin suppressed the enhanced renal gluconeogenesis enzymes and mitochondrial electron transport enzymes in type 2 diabetic rats and reduced the renal cytoplasmic glucose levels. The insulin index and pancreatic insulin granules were decreased in diabetic rats with increased V-ATPase expression in islet cells, and treatment with bafilomycin B1 reversed these changes and increased the insulin secretion index. Hepatosteatosis in type 2 diabetic rats was ameliorated by bafilomycin treatment. As a consequence, treatment with bafilomycin B1 significantly decreased the plasma glucose level after 24 h of starvation in diabetic rats. In conclusion, a V-ATPase inhibitor improved plasma glucose levels in type 2 diabetes by inhibiting renal mitochondrial gluconeogenesis and improving insulin secretion.

TIRC7 inhibits Th1 cells by upregulating the expression of CTLA­4 and STAT3 in mice with acute graft­versus­host disease.

In a previous study, it was demonstrated that T­cell immune response cDNA 7 (TIRC7) levels reflect the efficacy of treatment of patients with acute graft­versus­host disease (GVHD). However, the pathogenesis of TIRC7 in acute GVHD remains poorly understood. Lymphocytes from patients with acute GVHD were selected as targeT cells, and the effects of TIRC7 on cytotoxic T lymphocyte antigen­4 (CTLA­4), T cell activation and cytokine secretion were observed by electroporation. A mouse model of acute GVHD was established; anti­TIRC7 and anti­CTLA­4 monoclonal antibodies were intraperitoneally injected into recipient mice. Then, the effects of TIRC7 and CTLA­4 on T cell activation and acute GVHD were monitored. After TIRC7 expression was downregulated, CTLA­4 levels were decreased and STAT3 phosphorylation was reduced; conversely, the activation capacity of T lymphocytes was elevated, and the secretion of interferon­Î³ and other cytokines was increased. The mice in the TIRC7 + CTLA­4 co­administration group exhibited the lowest acute GVHD scores, with the longest average survival time and shortest recovery time of hematopoietic reconstitution. In conclusion, the results indicated that TIRC7 may positively regulate the function of CTLA­4 and inhibit T cell activation, thus suppressing the development and progression of acute GVHD.

Chloroquine and bafilomycin A mimic lysosomal storage disorders and impair mTORC1 signalling.

Autophagy is dependent upon lysosomes, which fuse with the autophagosome to complete the autophagic process and whose acidic interior permits the activity of their intraluminal degradative enzymes. Chloroquine (CQ) and bafilomycin A1 (BafA) each cause alkalinisation of the lumen and thus impair lysosomal function, although by distinct mechanisms. CQ diffuses into lysosomes and undergoes protonation, while BafA inhibits the ability of the vacuolar type H+-ATPase (v-ATPase) to transfer protons into the lysosome. In the present study, we examine the impact of CQ and BafA on the activity of mammalian target of rapamycin complex 1 (mTORC1), inhibition of which is an early step in promoting autophagy. We find each compound inhibits mTORC1 signalling, without affecting levels of protein components of the mTORC1 signalling pathway. Furthermore, these effects are not related to these agents' capacity to inhibit autophagy or the reduction in amino acid supply from lysosomal proteolysis. Instead, our data indicate that the reduction in mTORC1 signalling appears to be due to the accumulation of lysosomal storage material. However, there are differences in responses to these agents, for instance, in their abilities to up-regulate direct targets of transcription factor EB (TFEB), a substrate of mTORC1 that drives transcription of many lysosomal and autophagy-related genes. Nonetheless, our data imply that widely used agents that alkalinise intralysosomal pH are mimetics of acute lysosomal storage disorders (LSDs) and emphasise the importance of considering the result of CQ and BafA on mTORC1 signalling when interpreting the effects of these agents on cellular physiology.

The Vacuolar H+ ATPase α3 Subunit Negatively Regulates Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cells.

Increased metabolic acid production and upregulation of net acid extrusion render pH homeostasis profoundly dysregulated in many cancers. Plasma membrane activity of vacuolar H+ ATPases (V-ATPases) has been implicated in acid extrusion and invasiveness of some cancers, yet often on the basis of unspecific inhibitors. Serving as a membrane anchor directing V-ATPase localization, the a subunit of the V0 domain of the V-ATPase (ATP6V0a1-4) is particularly interesting in this regard. Here, we map the regulation and roles of ATP6V0a3 in migration, invasion, and growth in pancreatic ductal adenocarcinoma (PDAC) cells. a3 mRNA and protein levels were upregulated in PDAC cell lines compared to non-cancer pancreatic epithelial cells. Under control conditions, a3 localization was mainly endo-/lysosomal, and its knockdown had no detectable effect on pHi regulation after acid loading. V-ATPase inhibition, but not a3 knockdown, increased HIF-1 expression and decreased proliferation and autophagic flux under both starved and non-starved conditions, and spheroid growth of PDAC cells was also unaffected by a3 knockdown. Strikingly, a3 knockdown increased migration and transwell invasion of Panc-1 and BxPC-3 PDAC cells, and increased gelatin degradation in BxPC-3 cells yet decreased it in Panc-1 cells. We conclude that in these PDAC cells, a3 is upregulated and negatively regulates migration and invasion, likely in part via effects on extracellular matrix degradation.

Differential role of vacuolar (H+)-ATPase in the expression and activity of cyclooxygenase-2 in human monocytes.

Monocytes are professional immune cells that produce abundant levels of pro-inflammatory eicosanoids including prostaglandins and leukotrienes during inflammation. Vacuolar (H+)-ATPase (V-ATPase) is critically involved in a variety of inflammatory processes including cytokine trafficking and lipid mediator biosynthesis. However, its role in eicosanoid biosynthetic pathways in monocytes remains elusive. Here, we present a differential role of V-ATPase in the expression and in the activity of cyclooxygenase (COX)-2 in human monocytes. Pharmacological targeting of V-ATPase increased the expression of COX-2 protein in lipopolysaccharide-stimulated primary monocytes, which was paralleled by enhanced phosphorylation of p38 MAPK and ERK-1/2, without impacting the NF-κB and SAPK/JNK pathways. Targeting of both p38 MAPK and ERK-1/2 pathways showed that the kinase pathways are crucial for COX-2 expression in human monocytes. Despite increased COX-2 protein levels, however, suppression of V-ATPase activity impaired the biosynthesis of COX- and also of 5-lipoxygenase (LOX)-derived lipid mediators in monocytes without affecting 12-/15-LOX products, assessed by a metabololipidomics approach using UPLC-MS-MS analysis. Our results indicate that changes in the intracellular pH may contribute to suppression of COX-2 and 5-LOX activities. We suggest that V-ATPase on one hand limits COX-2 protein levels via restricting p38 MAPK and ERK-1/2 activation, while on the other hand it governs the cellular activity of COX-2 through appropriate adjustment of the intracellular pH.