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Vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases) are multi-component, ATP-driven proton pumps, which play important roles in many physiological processes by acidifying intracellular vesicles, organelles, and the extracellular milieu. Long-standing challenges in purifying mammalian V-ATPases have limited the biochemical and structural study of mammalian V-ATPase. Here, we provide a protocol for purifying milligrams of human V-ATPase and detail procedures for the reconstruction of its structure by cryo-EM. Our method can be applied to any biochemical and biophysical study of human V-ATPase. For complete details on the use and execution of this protocol, please refer to Wang et al. (2020)., Competing Interests: H.W. is a member of scientific advisory board of Cell. The other authors declare no competing interests., (© 2021 The Author(s).)

BACKGROUND The molecular mechanism of recurrent spontaneous abortion is unclear. It has been suggested that dysregulated genes participate in the pathogenesis of recurrent spontaneous abortion. The aim of this study was to identify the differentially expressed genes (DEGs) and pathways in recurrent spontaneous abortion. MATERIAL AND METHODS Gene expression data series of GSE22490 and GSE26787 were obtained from the GEO database to identify the differentially expressed genes between patients with recurrent miscarriage (Case group) and patients with uncomplicated pregnancies matched for gestational age (Control group). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEEG) were applied to enrich the biological functions and pathways of the identified differentially expressed genes. A protein-protein interaction (PPI) network was constructed thorough the STRING database. Thirty-one cases of recurrent spontaneous abortion (Case group) and 30 cases of artificial abortion (Control group) were included in the study. The protein expression of hub genes in the villi and decidua tissue of the 2 groups was detected by immunohistochemical assay. RESULTS Forty-six DEGs were identified with the enriched biological function mainly in the aspects of glutamate secretion and positive regulation of synapse assembly. KEGG pathway analysis indicated the dysregulated genes were only enriched in the glutamatergic synapse pathway. In the PPI network, 83 nodes and 273 edges with the average node degreed of 6.58 were enriched. The hub gene (ATP6V1G3) of the included 46 genes was identified using Cytohubba software. In the Case group, the high expression of ATP6V1G3 protein was detected in 13 (43.3%) and 10 (33.3%) for placental villus and decidual tissue, respectively. However, the high expression rate in the Control group was 23.3% and 16.7% for placental villus and decidual tissue, respectively. The ATP6V1G3 protein high expression rate was not significantly different between the Case and Control groups (P>0.05). CONCLUSIONS We found differential gene expression profiles in villous and decidual tissues between patients with recurrent miscarriage vs. those with uncomplicated pregnancies. Upregulation of the ATP6V1G3 gene may play an important role in the development of recurrent miscarriage.

V-ATPases are part of the membrane components of pathogen-containing vacuoles, although their function in intracellular infection remains elusive. In addition to organelle acidification, V-ATPases are alternatively implicated in membrane fusion and anti-inflammatory functions controlled by ATP6V0d2, the d subunit variant of the V-ATPase complex. Therefore, we evaluated the role of ATP6V0d2 in the biogenesis of pathogen-containing vacuoles using ATP6V0d2 knock-down macrophages infected with the protozoan parasite Leishmania amazonensis. These parasites survive within IFNγ/LPS-activated inflammatory macrophages, multiplying in large/fusogenic parasitophorous vacuoles (PVs) and inducing ATP6V0d2 upregulation. ATP6V0d2 knock-down decreased macrophage cholesterol levels and inhibited PV enlargement without interfering with parasite multiplication. However, parasites required ATP6V0d2 to resist the influx of oxidized low-density lipoprotein (ox-LDL)-derived cholesterol, which restored PV enlargement in ATP6V0d2 knock-down macrophages by replenishing macrophage cholesterol pools. Thus, we reveal parasite-mediated subversion of host V-ATPase function toward cholesterol retention, which is required for establishing an inflammation-resistant intracellular parasite niche., Competing Interests: The authors have declared that no competing interests exist.

Macrophages perform key functions in tissue homeostasis that are influenced by the local tissue environment. Within the tumor microenvironment, tumor-associated macrophages can be altered to acquire properties that enhance tumor growth. Here, we found that lactate, a metabolite found in high concentration within the anaerobic tumor environment, activated mTORC1 that subsequently suppressed TFEB-mediated expression of the macrophage-specific vacuolar ATPase subunit ATP6V0d2. Atp6v0d2-/- mice were more susceptible to tumor growth, with enhanced HIF-2α-mediated VEGF production in macrophages that display a more protumoral phenotype. We found that ATP6V0d2 targeted HIF-2α but not HIF-1α for lysosome-mediated degradation. Blockade of HIF-2α transcriptional activity reversed the susceptibility of Atp6v0d2-/- mice to tumor development. Furthermore, in a cohort of patients with lung adenocarcinoma, expression of ATP6V0d2 and HIF-2α was positively and negatively correlated with survival, respectively, suggesting a critical role of the macrophage lactate/ATP6V0d2/HIF-2α axis in maintaining tumor growth in human patients. Together, our results highlight the ability of tumor cells to modify the function of tumor-infiltrating macrophages to optimize the microenvironment for tumor growth.

Although individuals age and die with time, an animal species can continue indefinitely, because of its immortal germ-cell lineage. How the germline avoids transmitting damage from one generation to the next remains a fundamental question in biology. Here we identify a lysosomal switch that enhances germline proteostasis before fertilization. We find that Caenorhabditis elegans oocytes whose maturation is arrested by the absence of sperm exhibit hallmarks of proteostasis collapse, including protein aggregation. Remarkably, sperm-secreted hormones re-establish oocyte proteostasis once fertilization becomes imminent. Key to this restoration is activation of the vacuolar H + -ATPase (V-ATPase), a proton pump that acidifies lysosomes. Sperm stimulate V-ATPase activity in oocytes by signalling the degradation of GLD-1, a translational repressor that blocks V-ATPase synthesis. Activated lysosomes, in turn, promote a metabolic shift that mobilizes protein aggregates for degradation, and reset proteostasis by enveloping and clearing the aggregates. Lysosome acidification also occurs during Xenopus oocyte maturation; thus, a lysosomal switch that enhances oocyte proteostasis in anticipation of fertilization may be conserved in other species.

OBJECTIVE The (pro)renin receptor (PRR) plays an essential role in the early development of the central nervous system by activating the Wnt/β-catenin signaling pathway. The authors investigated the potential role of the PRR in the pathogenesis of glioma. METHODS The authors performed immunohistochemical analysis to detect both the PRR and isocitrate dehydrogenase 1 with mutations involving arginine 132 ( IDH1 R132H ) in paraffin sections of 31 gliomas. Expression of the PRR and Wnt pathway components in cultured human glioma cell lines (U251MG, U87MG, and T98G) was measured using Western blotting. The effects of PRR short interfering RNA (siRNA) on glioma cell proliferation (WST-1 assay and direct cell counting) and apoptosis (flow cytometry and the caspase-3 assay) were also examined. RESULTS PRR expression was significantly higher in glioblastoma than in normal tissue or in lower grade glioma, regardless of IDH1 R132H mutation. PRR expression was also higher in human glioblastoma cell lines than in human astrocytes. PRR expression showed a significant positive correlation with the Ki-67 labeling index, while it had a significant negative correlation with the survival time of glioma patients. Treatment with PRR siRNA significantly reduced expression of Wnt2, activated β-catenin, and cyclin D1 by human glioblastoma cell lines, and it reduced the proliferative capacity of these cell lines and induced apoptosis. CONCLUSIONS This is the first evidence that the PRR has an important role in development of glioma by aberrant activation of the Wnt/β-catenin signaling pathway. This receptor may be both a prognostic marker and a therapeutic target for glioma.

Recent studies demonstrate that the invasion and metastasis of gastric cancer (GC) is closely associated with a multi-subunit vacuolar H+-ATPase (V-ATPase). Here we investigated the expression and role of the human ATP6V1A gene that encodes the catalytic subunit A of V-ATPase in GC. We found that ATP6V1A expression level is significantly elevated in GCs compared to normals, but GC patients with higher expression levels of ATP6V1A have a better prognosis. Genomic analysis revealed that APT6V1A copy number is gained in a small fraction of GC patients and lost in a minimum number. Moreover, the ATP6V1A copy number was positively correlated with its mRNA level. To explore additional mechanisms by which ATP6V1A overexpressed in GCs, we investigated the relationship between transcription factor YY1 and ATP6V1A, and found that mRNA expression of YY1 had significant correlation with that of ATP6V1A. To validate that YY1 transcriptionally regulates ATP6V1A, we discovered that the ATP6V1A core promoter region contains three YY1 binding sites. Moreover, RNAi-mediated knockdown of YY1 in GC cells significantly decreased ATP6V1A mRNA and protein expression, while YY1 overexpression increased ATP6V1A expression level. In conclusion, YY1 may play an important regulatory role in ATP6V1A expression with potential mechanistic and clinical implications in GC.

Vps4, vacuolar protein sorting 4, belongs to ATPases Associated with diverse cellular Activities (AAA) protein family which is made up of Vps4A and Vps4B. Previous studies demonstrated that Vps4A plays vital roles in diverse aspects such as virus budding, the efficient transport of H-Ras to the PM (plasma membrane) and the involvement in the MVB (multivesiculate bodies) pathway. Interestingly, Vps4A is also expressed in the brain. However, the distribution and function of Vps4A in ICH diseases remain unclear. In this study, we show that Vps4A may be involved in neuronal apoptosis during pathophysiological processes of intracerebral hemorrhage (ICH). Based on the results of Western blot and immunohistochemistry, we found a remarkable up-regulation of Vps4A expression surrounding the hematoma after ICH. Double labeled immunofluorescence showed that Vps4A was co-expressed with NeuN but rarely with astrocytes and microglia. Morever, we detected that neuronal apoptosis marker active caspase-3 had co-localizations with Vps4A. Additionaly, Vps4A knockdown in vitro specifically leads to decreasing neuronal apoptosis coupled with increased Akt phosphorylation. All datas suggested that Vps4A was involved in promoting neuronal apoptosis via inhibiting Akt phosphorylation after ICH.

Desmodus rotundus is a vampire bat species that inhabits Latin America. Some basic aspects of this species' biology are still unknown, as the histophysiological characteristics of the male reproductive tract. Our study has focused on its epididymis, which is an important organ for performing a variety of functions, especially the sperm maturation and storage. The aim of this study was to identify principal, narrow, clear, and basal cells using cell-specific markers such as aquaporin 9 (AQP9), vacuolar H + -ATPase (V-ATPase), and cytokeratin 5 (KRT5). Principal cells were labeled by AQP9 from initial segment to cauda region in their stereocilia. They were shown with a columnar shape, whereas V-ATPase-rich cells were identified with a goblet-shaped body along the entire epididymis, including the initial segment, which were named as clear cells. Pencil-shaped V-ATPase-rich cells (narrow cells) were not detected in the initial segment of the bat epididymis, unlike in the rodent. Basal cells were labeled by KRT5 and were located at the basal portion of the epithelium forming a dense network. However, no basal cells with a luminal-reaching body extension were observed in the bat epididymis. In summary, epithelial cells were identified by their specific markers in the vampire bat epididymis. Principal and basal cells were labeled by AQP9 and KRT5, respectively. Narrow cells were not observed in the vampire bat epididymis, whereas clear cells were identified by V-ATPase labeling along the entire duct in a goblet-shaped body. In addition, no luminal-reaching basal cells were observed in the vampire bat epididymis.

Infantile malignant osteopetrosis (IMO) is a rare, recessive disorder characterized by increased bone mass caused by dysfunctional osteoclasts. The disease is most often caused by mutations in the TCIRG1 gene encoding a subunit of the V-ATPase involved in the osteoclasts capacity to resorb bone. We previously showed that osteoclast function can be restored by lentiviral vector-mediated expression of TCIRG1, but the exact threshold for restoration of resorption as well as the cellular response to vector-mediated TCIRG1 expression is unknown. Here we show that expression of TCIRG1 protein from a bicistronic TCIRG1/GFP lentiviral vector was only observed in mature osteoclasts, and not in their precursors or macrophages, in contrast to GFP expression, which was observed under all conditions. Thus, vector-mediated TCIRG1 expression appears to be post-transcriptionally regulated, preventing overexpression and/or ectopic expression and ensuring protein expression similar to that of wild-type osteoclasts. Codon optimization of TCIRG1 led to increased expression of mRNA but lower levels of protein and functional rescue. When assessing the functional rescue threshold in vitro, addition of 30 % CB CD34 + cells to IMO CD34 + patient cells was sufficient to completely normalize resorptive function after osteoclast differentiation. From both an efficacy and a safety perspective, these findings will clearly be of benefit during further development of gene therapy for osteopetrosis.

Although therapeutic hypothermia is an effective treatment for post-resuscitation brain injury after cardiac arrest (CA), the underlying mechanism remains unclear. Vacuolar H(+)-ATPase (V-ATPase) plays a key role in cellular adaption to a hypoxic environment. This study sought to evaluate the effect of mild hypothermia on V-ATPase and its involvement in neuroprotection after CA. Male Sprague-Dawley rats were subjected to a 6-min CA, resuscitated successfully, and then assigned to either the normothermia (NT) group or the hypothermia (HT) group. Rats were further divided into 2 subgroups based on the time of euthanasia, either 3 or 24 h after CA (NT-3 h, HT-3 h; NT-24 h, HT-24 h). Mild hypothermia was induced following CA and maintained at 33°C for 2 h. Neurologic deficit scores were used to determine the status of neurological function. Brain specimens were analyzed by TUNEL assay, western blotting, and immunohistochemistry. V-ATPase activity was estimated by subtracting total ATP hydrolysis from the bafilomycin-sensitive activity. Mild hypothermia improved the neurological outcome (HT-24 h: 34.3 ± 16.4 vs NT-24 h: 50.3 ± 17.4) and significantly decreased neurocyte apoptosis 24 h after resuscitation. Mild hypothermia significantly increased V0a1 compared to NT-3 h; V0a1 expression was associated with a decrease in the cleaved caspase 3 expression. These findings suggested that mild hypothermia inhibits CA-induced apoptosis in the hippocampus, which may be associated with reduced V-ATPase impairment. These data provide new insights into the protective effects of hypothermia in vivo.

To ascertain the epigenomic features, i.e., the methylation, non-coding RNA, and gene expression patterns, associated with gain of i(12p) in Pallister-Killian syndrome (PKS), we investigated single cell clones, harboring either disomy 12 or tetrasomy 12p, from a patient with PKS. The i(12p)-positive cells displayed a characteristic expression and methylation signature. Of all the genes on 12p, 13% were overexpressed, including the ATN1, COPS7A, and NECAP1 genes in 12p13.31, a region previously implicated in PKS. However, the median expression fold change (1.3) on 12p was lower than expected by tetrasomy 12p. Thus, partial dosage compensation occurs in cells with i(12p). The majority (89%) of the significantly deregulated genes were not situated on 12p, indicating that global perturbation of gene expression is a key pathogenetic event in PKS. Three genes-ATP6V1G1 in 9q32, GMPS in 3q25.31, and TBX5 in 12q24.21-exhibited concomitant hypermethylation and decreased expression. The i(12p)-positive cells displayed global hypomethylation of gene-poor regions on 12p, a footprint previously associated with constitutional and acquired gains of whole chromosomes as well as with X-chromosome inactivation in females. We hypothesize that this non-genic hypomethylation is associated with chromatin processing that facilitates cellular adaptation to excess genetic material.

Hearing impairment is the most frequent sensory deficit in humans. Deafness genes, which harbor pathogenic mutations that have been identified in families with hereditary hearing loss, are commonly expressed in the auditory end organ or the cochlea and may contribute to normal hearing function, yet some of the mouse models carrying these mutations fail to recapitulate the hearing loss phenotype. In this study, we find that distinct expression patterns of those deafness genes in the cochlea of a non-human primate, the common marmoset (Callithrix jacchus). We examined 20 genes whose expression in the cochlea has already been reported. The deafness genes GJB3, CRYM, GRHL2, DFNA5, and ATP6B1 were expressed in marmoset cochleae in patterns different from those in mouse cochleae. Of note, all those genes are causative for progressive hearing loss in humans, but not in mice. The other tested genes, including the deafness gene COCH, in which mutation recapitulates deafness in mice, were expressed in a similar manner in both species. The result suggests that the discrepancy in the expression between rodents and primates may account for the phenotypic difference. This limitation of the rodent models can be bypassed by using non-human primate models such as the marmoset.

The vacuolar-type H+ adenosine triphosphatase (V-ATPase) plays an important role in cellular acidification and bone resorption by osteoclasts. However, the direct effect of bovine parathyroid hormone (bPTH) on V-ATPase has not yet been elucidated. The aim of the present study was to assess the effects of bPTH on V-ATPase and osteoclasts. Osteoclasts from bone marrow (BM)-derived monocytes of C57BL/6 mice were cultured with or without bPTH. The mRNA and protein expression levels of the V-ATPase a3-subunit and d2-subunit (by RT-qPCR and western blot analysis), V-ATPase activity (using the V type ATPase Activity Assay kit) and the bone resorption function of osteoclasts (by bone resorption assay) were examined following treatment with various concentrations of bPTH (0.1, 1.0, 10 and 100 ng/ml) alone or with bPTH and its inhibitor, bafilomycin A1. Furthermore, the expression of parathyroid hormone (PTH) receptors in osteoclasts was also detected. The results revealed that the mRNA and protein expression levels of V-ATPase a3-subunit and d2-subunit increased in a dose‑dependent manner, paralleling the level of bPTH present. In addition, an increase in the concentration of bPTH was accompanied by the increased resorption capability of osteoclasts, whereas bone resorption was inhibited in the presence of bafilomycin A1. In addition, we confirmed the existence of parathyroid hormone 1 receptor (PTH1R) in osteoclasts using three different methods (RT-qPCR, western blot analysis and immunofluorescence staining). We found that bPTH enhanced the bone resorption capability of osteoclasts by modulating the expression of V-ATPase subunits, intracellular acidification and V-ATPase activity. Thus, we propose that PTH has a direct effect on osteoblasts and osteoclasts, and that this effect is mediated through PTH1R, thus contributing to bone remodeling.

Insulin is one of the main factors affecting bone and energy metabolism, however, the direct effect of insulin on osteoclast differentiation remains unclear. Thus, in order to help elucidate that puzzle, the authors investigated the roles and regulatory mechanisms of insulin on osteoclasts differentiation. Co-stimulation with insulin and RANKL significantly enhanced the number of larger (>100 μm) osteoclastic cells and of TRAP-positive multinucleated cells compared with treatment by RANKL alone. Conversely, the insulin receptor shRNA markedly decreased osteoclast differentiation induced by insulin and RANKL. Insulin treatment significantly activated ERK1/2 MAP kinase as well as markedly induced the expression of NFATc1, an osteoclast marker gene, and Atp6v0d2, an osteoclast fusion-related gene. The pretreatment of PD98059, an ERK1/2 inhibitor, or insulin receptor shRNA effectively suppressed osteoclast differentiation and, in addition, blocked the expression of NFATc1 and Atp6vod2 induced by insulin stimulation. These data reveal insights into the regulation of osteoclast differentiation and fusion through ERK1/2 activation and the induction of NFATc1 and Atp6v0d2 by insulin., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Autosomal recessive osteopetrosis (ARO) is a rare genetic bone disease with genotypic and phenotypic heterogeneity, sometimes translating into delayed diagnosis and treatment. In particular, cases of intermediate severity often constitute a diagnostic challenge and represent good candidates for exome sequencing. Here, we describe the tortuous path to identification of the molecular defect in two siblings, in which osteopetrosis diagnosed in early childhood followed a milder course, allowing them to reach the adult age in relatively good conditions with no specific therapy. No clearly pathogenic mutation was identified either with standard amplification and resequencing protocols or with exome sequencing analysis. While evaluating the possible impact of a 3'UTR variant on the TCIRG1 expression, we found a novel single nucleotide change buried in the middle of intron 15 of the TCIRG1 gene, about 150 nucleotides away from the closest canonical splice site. By sequencing a number of independent cDNA clones covering exons 14 to 17, we demonstrated that this mutation reduced splicing efficiency but did not completely abrogate the production of the normal transcript. Prompted by this finding, we sequenced the same genomic region in 33 patients from our unresolved ARO cohort and found three additional novel single nucleotide changes in a similar location and with a predicted disruptive effect on splicing, further confirmed in one of them at the transcript level. Overall, we identified an intronic region in TCIRG1 that seems to be particularly prone to splicing mutations, allowing the production of a small amount of protein sufficient to reduce the severity of the phenotype usually associated with TCIRG1 defects. On this basis, we would recommend including TCIRG1 not only in the molecular work-up of severe infantile osteopetrosis but also in intermediate cases and carefully evaluating the possible effects of intronic changes., (© 2015 American Society for Bone and Mineral Research.)

The hormonally active form of vitamin D3, 1α,25-(OH)2D3, has an important role in bone metabolism. This study examined the effects of 1α,25-(OH)2D3 on the ability of two cytokines, receptor activator of nuclear factor-κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), to induce RAW 264.7 cells to form osteoclasts. A TRAP histochemical staining assay and bone resorption analysis were used to identify the rate of formation and activity of osteoclasts. The numbers of osteoclasts formed, and their bone resorption activity, was enhanced by the addition of 1α,25-(OH)2D3. The expression levels of osteoclast-specific proteins that are essential for bone resorption, integrin β3, V-ATPase, CAII, CTSK, TRAP and MMP-9, were detected by western blotting. During 48 h, the expression levels of all these proteins significantly increased. Quantitative real-time polymerase chain reaction was used to determine the expression levels of the transcription factors, c-Fos and NFATcl. The expression levels of c-Fos and NFATc1 also increased 24h after treatment with 1α,25-(OH)2D3. These results suggest that 1α,25-(OH)2D3 can regulate bone metabolism by directly enhancing the formation and maturation of osteoclasts., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Acute graft-versus-host disease (aGVHD) has become the important complication post-allogeneic hematopoietic stem cell transplantation. Abnormally activated T cells might play an important role in the pathogenesis of aGVHD. But its exact mechanism remains poorly understood. T cell immune response cDNA 7 (TIRC7) has been identified to be essential in T cell activation; however, the role of TIRC7 in aGVHD remains unclear. The purpose of this study was to measure the expression of TIRC7 and T helper (Th) cells in patients with aGVHD before and after treatment. We showed that TIRC7 levels in aGVHD patients were higher than those of healthy controls and markedly declined after treatment. The levels of IFN-γ (Th1), IL-17 (Th17), and IL-22 (Th22) were in accordance with the grade of aGVHD. In addition, TIRC7 levels were also associated with the severity of aGVHD. In conclusion, TIRC7 might be involved in the pathogenesis of aGVHD and TIRC7 level might be an indicator to evaluate the response of patients with aGVHD to treatment.

Differentiating between chromophobe renal cell carcinoma (RCC) and other RCC subtypes can be problematic using routine light microscopy. This study aimed to identify novel immunohistochemical markers useful for a differential diagnosis between chromophobe RCC and other RCC subtypes. We selected 3 genes (including BSND and ATP6V1G3) that showed specific transcriptional expression in chromophobe RCC using expression data (n = 783) from The Cancer Genome Atlas (TCGA) database. A subsequent immunohistochemical examination of 186 RCCs obtained in our patient series resulted in a strong diffuse positivity of BSND and ATP6V1G3 proteins (both of which are involved in the regulation of membrane transport) in all the chromophobe RCC specimens (23/23 cases, 100%) but not in the clear cell RCC specimens (0/153 cases, 0%) or the papillary RCC specimens (0/10 cases, 0%). BSND and ATP6V1G3 protein expressions were also detected in renal oncocytoma (13/14 cases, 92.9%) and in the distal nephron, including the collecting duct, in the normal kidney. A computational analysis of TCGA data suggested that DNA methylation was involved in the differential expression pattern of both genes among RCC subtypes. Finally, an immunohistochemical analysis showed lung carcinomas were negative (0/85 cases, 0%) for the expression of both proteins. These results suggest that BSND and ATP6V1G3 are excellent novel immunohistochemical markers for differentiating between chromophobe RCC and other subtypes of RCC, including clear cell and papillary RCCs.

Background: Atrial fibrillation (AF) is the most common cardiac arrhythmia, causing substantial cardiovascular morbidity and mortality. The renin-angiotensin system (RAS) has been shown to be involved in the pathophysiology of AF. The (pro)renin receptor [(p)RR] is the last identified member of RAS. However, the role of (p)RR in AF is still unknown., Methods and Results: Circulating levels of (p)RR were determined using an immunosorbent assay in 22 patients with AF (paroxysmal or persistent) and 22 healthy individuals. The plasma levels of (p)RR increased 3.6-fold in AF patients (P<0.001), indicating a relationship between (p)RR and AF. To investigate the role of (p)RR in the regulation of cardiac arrhythmia, we generated a transgenic mouse with overexpression of human (p)RR gene specifically in the heart. Electrocardiograms from (p)RR transgenic mice showed typical atrial flutter since 2 months, then spontaneously converted to atrial fibrillation by 10 months. The atria of the transgenic mice demonstrated significant dilation and fibrosis, and exhibited a high incidence of sudden death. Additionally, the genes of SERCA and HCN4, which are involved in the electrophysiology of AF, were significantly down-regulated and up-regulated respectively in transgenic mice atria. The phosphorylation of Erk1/2 significantly increased in the atria of the transgenic mice, and the activated Erk1/2 was found predominantly in cardiac fibroblasts, suggesting that the transgenic (p)RR gene may induce atrial fibrillation by activation of Erk1/2 in the cardiac fibroblasts of the atria., Conclusions: (p)RR promotes atrial structural and electrical remodeling in vivo, which indicates that (p)RR plays an important role in the pathological development of AF., (Copyright © 2015. Published by Elsevier Ireland Ltd.)

Homo sapiens longevity assurance homologue 2 of yeast LAG1 (LASS2)/tumor metastasis suppressor gene 1 (TMSG1) was a novel tumor metastasis-related gene identified using messenger RNA differential display from non-metastatic human prostate cancer cell variants. The mechanism of LASS2/TMSG1 inhibiting tumor invasion metastasis in breast cancer cells had not been well investigated. In the present study, a full length of 1.2 kb LASS2/TMSG1 complementary DNA (cDNA) coding for a protein of 380 amino acids was cloned. PcDNA3 eukaryotic expression plasmids of LASS2/TMSG1 were constructed and transfected into human breast cancer cell line MCF-7 by lipofectin transfection method. And, the biological effects were observed comparing with control groups. As the result, LASS2/TMSG1 inhibited cell growth in vitro by increasing apoptosis and changing cell cycle distribution. Furthermore, the vacuolar ATPase (V-ATPase) activity and extracellular hydrogen ion concentration were significantly decreased and the activity of secreted matrix metalloproteinase-2 (MMP-2) was downregulated in MCF-7 cells overexpressing LASS2/TMSG1 compared with the controls. Therefore, LASS2/TMSG1 may inhibit growth and invasion of breast cancer cell in vitro through decreasing V-ATPase activity and extracellular hydrogen ion concentration and inactivating secreted MMP-2. The findings provided the evidence that the LASS2/TMSG1 gene had tumor growth and invasion suppressor function in human breast cancer cell and may provide a promising target for cancer metastasis diagnosis and therapy.

Purpose: Extranodal marginal zone B-cell lymphoma (EMZL) is the most common subtype of conjunctival lymphoma, though its molecular mechanisms of pathogenesis are largely unknown. We attempted to explore the association of the renin-angiotensin system (RAS) and (pro)renin receptor ([P]RR) in the pathogenesis of conjunctival lymphoma., Methods: Surgically removed conjunctiva EMZL samples were used for gene expression, and immunohistochemical and immunofluorescence analyses of (P)RR and RAS components. Human B-lymphoblast IM-9 cells were treated with prorenin or angiotensin II (Ang II), and gene expression levels were analyzed using real-time quantitative PCR (qPCR). In addition, immunofluorescence analysis of EMZL samples was used to evaluate the in vivo expression of those components., Results: Gene expression and immunohistochemical analyses revealed the expression of RAS components, including (P)RR and angiotensin II type 1 receptor (AT1R), in EMZL tissues. Double-labeling analyses demonstrated that (P)RR and AT1R were detected in cells positive for CD20, a marker for B-cells, where they colocalized with prorenin and angiotensinogen, respectively. Prorenin stimulation of human B-lymphoblast IM-9 cells increased mRNA expression levels of fibroblast growth factor 2 (FGF2), while angiotensin II treatment upregulated the expression levels of basigin (BSG), matrix metallopeptidase (MMP)2, 9, and 14, which were abolished by (P)RR and AT1R blockades, respectively. Immunofluorescence analyses of clinical samples showed colocalizations of (P)RR and AT1R with the products of these upregulated genes., Conclusions: The present study suggests that activation of (P)RR and AT1R is associated with the pathogenesis of conjunctival EMZL by stimulating the production of FGF2 and MMPs., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Unlabelled: Chronic kidney disease (CKD) is associated with an increased risk of cardiovascular disease (CVD). (Pro)renin receptor (PRR) is activated in the kidney of CKD. The present study aimed to determine the role of indoxyl sulfate (IS), a uremic toxin, in PRR activation in rat aorta and human aortic smooth muscle cells (HASMCs). We examined the expression of PRR and renin/prorenin in rat aorta using immunohistochemistry. Both CKD rats and IS-administrated rats showed elevated expression of PRR and renin/prorenin in aorta compared with normal rats. IS upregulated the expression of PRR and prorenin in HASMCs. N-acetylcysteine, an antioxidant, and diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, suppressed IS-induced expression of PRR and prorenin in HASMCs. Knock down of organic anion transporter 3 (OAT3), aryl hydrocarbon receptor (AhR) and nuclear factor-κB p65 (NF-κB p65) with small interfering RNAs inhibited IS-induced expression of PRR and prorenin in HASMCs. Knock down of PRR inhibited cell proliferation and tissue factor expression induced by not only prorenin but also IS in HASMCs., Conclusion: IS stimulates aortic expression of PRR and renin/prorenin through OAT3-mediated uptake, production of reactive oxygen species, and activation of AhR and NF-κB p65 in vascular smooth muscle cells. IS-induced activation of PRR promotes cell proliferation and tissue factor expression in vascular smooth muscle cells.

Rationale: The rationale was to utilize a bioinformatics approach to identify miRNA binding sites in genes with single nucleotide mutations (SNPs) to discover pathways in heart failure (HF)., Objective: The objective was to focus on the genes containing miRNA binding sites with miRNAs that were significantly altered in end-stage HF and in response to a left ventricular assist device (LVAD)., Methods and Results: BEDTools v2.14.3 was used to discriminate SNPs within predicted 3'UTR miRNA binding sites. A member of the miR-15/107 family, miR-16, was decreased in the circulation of end-stage HF patients and increased in response to a LVAD (p<0.001). MiR-16 decreased Vacuolar Protein Sorting 4a (VPS4a) expression in HEK 293T cells (p<0.01). The SNP rs16958754 was identified in the miR-15/107 family binding site of VPS4a which abolished direct binding of miR-16 to the 3'UTR of VPS4a (p<0.05). VPS4a was increased in the circulation of end-stage HF patients (p<0.001), and led to a decrease in the number of HEK 293T cells in vitro (p<0.001)., Conclusions: We provide evidence that miR-16 decreases in the circulation of end-stage HF patients and increases with a LVAD. Modeling studies suggest that miR-16 binds to and decreases expression of VPS4a. Overexpression of VPS4a decreases cell number. Together, these experiments suggest that miR-16 and VPS4a expression are altered in end-stage HF and in response to unloading with a LVAD. This signaling pathway may lead to reduced circulating cell number in HF.

Vacuolar proton-translocating ATPases (V-ATPases) are highly conserved, ATP-driven proton pumps regulated by reversible dissociation of its cytosolic, peripheral V1 domain from the integral membrane V(o) domain. Multiple stresses induce changes in V1-V(o) assembly, but the signaling mechanisms behind these changes are not understood. Here we show that certain stress-responsive changes in V-ATPase activity and assembly require the signaling lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). V-ATPase activation through V1-V(o) assembly in response to salt stress is strongly dependent on PI(3,5)P2 synthesis. Purified V(o) complexes preferentially bind to PI(3,5)P2 on lipid arrays, suggesting direct binding between the lipid and the membrane sector of the V-ATPase. Increasing PI(3,5)P2 levels in vivo recruits the N-terminal domain of V(o)-sector subunit Vph1p from cytosol to membranes, independent of other subunits. This Vph1p domain is critical for V1-V(o) interaction, suggesting that interaction of Vph1p with PI(3,5)P2-containing membranes stabilizes V1-V(o) assembly and thus increases V-ATPase activity. These results help explain the previously described vacuolar acidification defect in yeast fab1 and vac14 mutants and suggest that human disease phenotypes associated with PI(3,5)P2 loss may arise from compromised V-ATPase stability and regulation.

Physiological and proteomic responses of Sesuvium portulacastrum leaves under salinity were investigated. Different from glycophytes, this halophyte had optimal growth at 200-300mM NaCl and accumulated more starch grains in chloroplasts under high salinity. Increased contents of soluble sugars, proline, and Na(+) were observed upon salinity. X-ray microanalysis revealed that Na(+) was mainly compartmentalized into cell vacuole. Quantitative proteomics produced 96 salt responsive proteins, and the majority was chloroplast-located proteins. Gene ontology analysis revealed that proteins involved in ion binding, proton transport, photosynthesis and ATP synthesis were overrepresented. The expressions of a Na(+)/H(+) antiporter and several ATP synthase subunits were activated upon high salinity. ATP hydrolysis assay demonstrated that V-ATPase activity at tonoplast was dramatically increased upon NaCl whereas vacuolar H(+)-pyrophosphatase and plasma membrane P-ATPase activities were not increased, which indicated that sodium compartmentalization was mainly performed by enhancing V-ATPase activity rather than P-ATPase and H(+)-pyrophosphatase. Accumulation of soluble sugars as well as sodium compartmentalization maintained the osmotic balance between vacuole and cytoplasm, which finally established ionic homeostasis in saline cells in true halophytes., Biological Significance: Physiological and proteomic analyses of S. portulacastrum leaves under different salinities were investigated. This true halophyte accumulated more soluble sugars, starch, proline and Na(+) under high salinity. Differential proteomics produced 96 salt responsive proteins and the majority was involved in ion binding, proton transport, photosynthesis, and ATP synthesis. A Na(+)/H(+) antiporter and several ATP synthase subunits were induced upon high salinity. ATP hydrolysis assay demonstrated that V-ATPase activity at tonoplast was dramatically increased whereas vacuolar H(+)-pyrophosphatase and plasma membrane ATPase activities were stable upon NaCl. These findings demonstrated that the increased Na(+) was compartmentalized into vacuole by enhancing V-ATPase activity rather than H(+)-ATPase., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

Background: Hyperbaric oxygenation was shown to increase bone healing in a rabbit model. However, little is known about the regulatory factors and molecular mechanism involved.We hypothesized that the effect of hyperbaric oxygen (HBO) on bone formation is mediated via increases in the osteogenic differentiation of mesenchymal stem cells (MSCs) which are regulated by Wnt signaling., Methods: The phenotypic characterization of the MSCs was analyzed by flow cytometric analysis. To investigate the effects of HBO on Wnt signaling and osteogenic differentiation of MSCs, mRNA and protein levels of Wnt3a, beta-catenin, GSK-3beta, Runx 2, as well as alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining were analyzed after HBO treatment. To investigate the effects of HBO on Wnt processing and secretion, the expression of Wntless and vacuolar ATPases were quantified after HBO treatment., Results: Cells expressed MSC markers such as CD105, CD146, and STRO-1. The mRNA and protein levels of Wnt3a, β-catenin, and Runx 2 were up-regulated, while GSK-3β was down-regulated after HBO treatment. Western blot analysis showed an increased β-catenin translocation with a subsequent stimulation of the expression of target genes after HBO treatment. The above observation was confirmed by small interfering (si)RNA treatment. HBO significantly increased alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining of osteogenically differentiated MSCs. We further showed that HBO treatment increased the expression of Wntless, a retromer trafficking protein, and vacuolar ATPases to stimulate Wnt processing and secretion, and the effect was confirmed by siRNA treatment., Conclusions: HBO treatment increased osteogenic differentiation of MSCs via regulating Wnt processing, secretion, and signaling.

The regulator of ATPase of vacuoles and endosomes (RAVE) complex is implicated in vacuolar H(+)-translocating ATPase (V-ATPase) assembly and activity. In yeast, rav1 mutants exhibit a Vma(-) growth phenotype characteristic of loss of V-ATPase activity only at high temperature. Synthetic genetic analysis identified mutations that exhibit a full, temperature-independent Vma(-) growth defect when combined with the rav1 mutation. These include class E vps mutations, which compromise endosomal sorting. The synthetic Vma(-) growth defect could not be attributed to loss of vacuolar acidification in the double mutants, as there was no vacuolar acidification in the rav1 mutant. The yeast V-ATPase a subunit is present as two isoforms, Stv1p in Golgi and endosomes and Vph1p in vacuoles. Rav1p interacts directly with the N-terminal domain of Vph1p. STV1 overexpression suppressed the growth defects of both rav1 and rav1vph1, and allowed RAVE-independent assembly of active Stv1p-containing V-ATPases in vacuoles. Mutations causing synthetic genetic defects in combination with rav1 perturbed the normal localization of Stv1-green fluorescent protein. We propose that RAVE is necessary for assembly of Vph1-containing V-ATPase complexes but not Stv1-containing complexes. Synthetic Vma(-) phenotypes arise from defects in Vph1p-containing complexes caused by rav1, combined with defects in Stv1p-containing V-ATPases caused by the second mutation. Thus RAVE is the first isoform-specific V-ATPase assembly factor.

Vacuolar (V)-ATPase is a proton-translocating enzyme that acidifies cellular compartments for various functions such as receptor-mediated endocytosis, intracellular trafficking and protein degradation. Previous studies in Dermacentor variabilis chronically infected with Rickettsia montanensis have identified V-ATPase as one of the tick-derived molecules transcribed in response to rickettsial infection. To examine the role of the tick V-ATPase in tick-Rickettsia interactions, a full-length 2887-bp cDNA (2532-bp open reading frame) clone corresponding to the transcript of the V0 domain subunit a of D. variabilis V-ATPase (DvVATPaseV0a) gene encoding an 843 amino acid protein with an estimated molecular weight of ~96 kDa was isolated from D. variabilis. Amino acid sequence analysis of DvVATPaseV0a showed the highest similarity to VATPaseV0a from Ixodes scapularis. A potential N-glycosylation site and eight putative transmembrane segments were identified in the sequence. Western blot analysis of tick tissues probed with polyclonal antibody raised against recombinant DvVATPaseV0a revealed the expression of V-ATPase in the tick ovary. Transcriptional profiles of DvVATPaseV0a demonstrated a greater mRNA expression in the tick ovary, compared with the midgut and salivary glands; however, the mRNA level in each of these tick tissues remained unchanged after infection with R. montanensis for 1 h. V-ATPase inhibition bioassays resulted in a significant decrease in the ability of R. montanensis to invade tick cells in vitro, suggesting a role of V-ATPase in rickettsial infection of tick cells. Characterization of tick-derived molecules involved in rickettsial infection is essential for a thorough understanding of rickettsial transmission within tick populations and the ecology of tick-borne rickettsial diseases., (© 2013 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society.)

Genes for V-H(+)-ATPase subunits were identified and cloned from the salt-tolerant wheat mutant RH8706-49. Sequences of these genes are highly conserved in plants. Overexpression of these genes in Arabidopsis thaliana improved its salt tolerance, and increased the activities of V-H(+)-ATPase and Na(+)/H(+) exchange, with the largest increase in plants carrying the c subunit of V-H(+)-ATPase. Results from quantitative RT-PCR analysis indicated that the mRNA level of each V-H(+)-ATPase subunit in the Arabidopsis increased under salt stress. Overall, our results suggest that each V-H(+)-ATPase subunit plays a key role in enhancing salt tolerance in plants.

Homo sapiens longevity assurance homologue 2 of yeast LAG1 (LASS2), also known as tumor metastasis suppressor gene 1 (TMSG1), is a newly found tumor metastasis suppressor gene in 1999. Preliminary studies showed that it not only suppressed tumor growth but also closely related to tumor metastasis, however, its molecular mechanisms is still unclear. There have been reported that protein encoded by LASS2/TMSG-1 could directly interact with the C subunit of Vacuolar ATPase (V-ATPase), which suggested that LASS2/TMSG1 might inhibit the invasion and metastasis through regulating the function of V-ATPase. Thus, in this study, we explored the effect of small interference RNA (siRNA) targeting LASS2/TMSG1 on the invasion of human prostate carcinoma cell line PC-3M-2B4 and its molecular mechanisms associated with the V-ATPase. Real-time fluorogentic quantitative PCR (RFQ-PCR) and Western blot revealed dramatic reduction of 84.5% and 60% in the levels of LASS2/TMSG1 mRNA and protein after transfection of siRNA in PC-3M-2B4 cells. The V-ATPase activity and extracellular hydrogen ion concentration were significantly increased in 2B4 cells transfected with the LASS2/TMSG1-siRNA compared with the controls. The activity of secreted MMP-2 was up-regulated in LASS2/TMSG1-siRNA treated cells compared with the controls; and the capacity for migration and invasion in LASS2/TMSG1-siRNA treated cells was significantly higher than the controls. Thus, we concluded that silencing of LASS2/TMSG1 may promote invasion of prostate cancer cell in vitro through increase of V-ATPase activity and extracellular hydrogen ion concentration and in turn the activation of secreted MMP-2., (Copyright © 2012 Wiley Periodicals, Inc.)

The renin-angiotensin system (RAS) is thought to regulate placentation, however, the expression and localization of RAS pathways in early gestation human placenta is not known. Here we describe the expression of prorenin (REN), (pro)renin receptor (ATP6AP2), angiotensinogen (AGT), angiotensin-converting enzyme 1 and 2 (ACE; ACE2), angiotensin II type 1 and 2 receptors (AGTR1; AGTR2) and angiotensin 1-7 receptor (MAS1), as well as the angiogenic factor, vascular endothelial growth factor (VEGF), and transforming growth factor-β1 (TGF-β1), in early gestation (6-16 weeks) and term (>37 weeks) human placentae. We also describe the location of all of the key RAS proteins in the early gestation placentae. The highest levels of REN, ATP6AP2, AGT, AGTR1 and ACE2 mRNAs were found in early gestation, whereas ACE1 mRNA was highest at term. AGTR2 and MAS1 mRNA expression were low to undetectable in all samples. REN, ATP6AP2 and AGTR1 mRNA levels were correlated with VEGF expression, but not with TGF-β1 mRNA. In early gestation placentae, prorenin, (pro)renin receptor and the angiotensin II type 1 receptor (AT(1)R) were localized to extravillous trophoblast cells, suggesting they play a key role in trophoblast migration. ACE2 in syncytiotrophoblasts could regulate release of Ang 1-7 into the maternal circulation contributing to the vasodilation of the maternal vasculature. ACE was only found in fetal vascular endothelium and may specifically target the growing fetal placental vessels. Because REN, ATP6AP2 and AGTR1 show strong correlations with expression of VEGF this pathway is likely to be important in placental angiogenesis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Iron is essential for all living organisms and plays a crucial role in pathogenicity. This study presents the first proteome analysis of plasma membranes isolated from pea roots. Protein profiles of four different samples (+Fe, +Fe/Chitosan, -Fe, and -Fe/Chitosan) were compared by native IEF-PAGE combined with in-gel activity stains and DIGE. Using DIGE, 89 proteins of interest were detected in plasma membrane fractions. Data revealed a differential abundance of several spots in all samples investigated. In comparison to the control and -FeCh the abundance of six protein spots increased whereas 56 spots decreased in +FeCh. Altered protein spots were analyzed by MALDI-TOF-TOF mass spectrometry. Besides stress-related proteins, transport proteins and redox enzymes were identified. Activity stains after native PAGE and spectrophotometric measurements demonstrated induction of a ferric-chelate reductase (-Fe) and a putative respiratory burst oxidase homolog (-FeCh). However, the activity of the ferric-chelate reductase decreased in -Fe plants after elicitor treatment. The activity of plasma membrane-bound class III peroxidases increased after elicitor treatment and decreased under iron-deficiency, whereas activity of quinone reductases decreased mostly after elicitor treatment. Possible functions of proteins identified and reasons for a weakened pathogen response of iron-deficient plants were discussed., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is an important, highly conserved, regulator of cell growth. Ancient among the signals that regulate mTORC1 are nutrients. Amino acids direct mTORC1 to the surface of the late endosome/lysosome, where mTORC1 becomes receptive to other inputs. However, the interplay between endosomes and mTORC1 is poorly understood. Here, we report the discovery of a network that links mTORC1 to a critical component of the late endosome/lysosome, the V-ATPase. In an unbiased screen, we found that mTORC1 regulated the expression of, among other lysosomal genes, the V-ATPases. mTORC1 regulates V-ATPase expression both in cells and in mice. V-ATPase regulation by mTORC1 involves a transcription factor translocated in renal cancer, TFEB. TFEB is required for the expression of a large subset of mTORC1 responsive genes. mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization and in a manner dependent on both TFEB and V-ATPases, mTORC1 promotes endocytosis. These data uncover a regulatory network linking an oncogenic transcription factor that is a master regulator of lysosomal biogenesis, TFEB, to mTORC1 and endocytosis.

Purpose: Osteonecrosis of the jaw (ONJ) is a growing concern in patients who receive bisphosphonates that target osteoclasts. As osteoclasts play multifunctional roles in the bone marrow, their suppression likely affects bone homeostasis and alters wound healing of the jaw. The objective was to delineate the impact of osteoclast suppression in the bone marrow and wound healing of the jaw., Experimental Design: Zoledronate was administered to senile rats for 14 weeks. A portion of the gingiva was removed to denude the palatal bone. Gene expression in the bone marrow was assessed and histologic sections were analyzed to determine the wound healing status., Results: Angiogenesis-related genes, CD31 and VEGF-A, were not altered by zoledronate. VEGF-C, which plays a role in lymphangiogenesis, was suppressed. There was a decrease in gene expression of Tcirg1 and MMP-13. Bone denudation caused extensive osteocyte death indicative of bone necrosis. In zoledronate-treated rats, the necrotic bone was retained in the wound while, in controls, osteoclastic resorption of the necrotic bone was prominent. Even though large necrotic bone areas existed in zoledronate-treated rats, overlaying soft tissue healed clinically. Immunohistochemical staining showed rich vascularity in the overlaying soft tissue., Conclusions: Zoledronate therapy impacts bone marrow by suppressing genes associated with lymphangiogenesis and tissue remodeling, such as VEGF-C and MMP-13. Zoledronate was associated with impaired osseous wound healing but had no effect on angiogenic markers in the bone marrow or soft tissue wound healing. Zoledronate selectively blunts healing in bone but does not affect soft tissue healing in the oral cavity., (©2010 AACR.)

Climate change affects organisms that inhabit not only in aerial but also in aquatic environments by making water more hypoxic and acidic. In the past, we evaluated morphological and functional variations in the gills of 12 species of aquatic air-breathing fishes. The aim of the present study is to examine the degree of gill modification in the aquatic air-breathing fish, Trichogaster lalius, in response to acidic stress. This provides a link between the ecological and physiological studies. We evaluated the changes in morphology and function of the gills, labyrinth organ, and kidney when the fish were subjected to acidic water and deionized water (DW). In the first experiment, fish were sampled at 1, 2, 4, and 7 days after acidic treatment. Apparent morphological modification was observed on day 4 and recovery was noted on day 7. Protein expression and enzyme activity of vacuolar-type H+-ATPase (VHA) and the protein expression of the proliferating cell nuclear antigen (PCNA) of the 1st and 4th gill arches both increased in the 4-day and 7-day acidic groups while the enzyme activity of Na+/K+-ATPase (NKA) decreased. In the second experiment, fish were tested for changes in the 1st and 4th gill arches and kidney after exposure to DW and acidic water for 4days. The gill structure of the fish in the DW was not different from that of the control group (fresh water). The protein expression and enzyme activity of the VHA of the 1st and 4th gill arches increased in both the DW and acidic groups for 4 days. We found a decrease in the protein expression of NKA in the kidney and in the enzyme activity of NKA in the 1st and 4th gill arches in the DW and acidic groups. From these results, we suggest that T. lalius exhibited significantly different ionic regulation and acid-base regulatory abilities in the DW and acidic groups in the 1st and 4th gill arches and kidney. The responses of the gills in T. lalius were different from those fish that show apparent morphological variations between the 1st and 4th gill arches., (Crown Copyright © 2010. Published by Elsevier Inc. All rights reserved.)

Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for Saccharomyces cerevisiae survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63. hph1Δ hph2Δ cells display reduced vacuole acidification; increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase); and growth defects similar to those of mutants lacking V-ATPase activity. sec71Δ cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in hph1Δ hph2Δ and hph1Δ hph2Δ sec71Δ cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates hph1Δ hph2Δ phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.

Objective: The aim of this study was to assess the effect of cigarette smoke inhalation (CSI) on gene expression in alveolar bone healing sites., Study Design: Wistar rats were randomly assigned to the groups: control [animals not exposed to CSI (n = 20)] and test [animals exposed to CSI, starting 3 days before teeth extraction and maintained until killing them (n = 20)]. First mandibular molars were bilaterally extracted, and the expression of alkaline phosphatase, bone morphogenetic protein (BMP) 2 and 7, receptor activator of nuclear factor κB ligand, osteoprotegerin, and d2 isoform of vacuolar adenosine triphosphatase V(0) domain were assessed by quantitative polymerase chain reaction in the newly formed tissue in the sockets., Results: Overall, data analysis demonstrated that CSI significantly affected the expression pattern of all of the studied genes except BMP-7., Conclusion: The expression of key genes for bone healing may be affected by CSI in tooth extraction sites., (Copyright © 2010 Mosby, Inc. All rights reserved.)

How individual protein subunits assemble into the higher order structure of a protein complex is not well understood. Four proteins dedicated to the assembly of the V(0) subcomplex of the V-adenosine triphosphatase (V-ATPase) in the endoplasmic reticulum (ER) have been identified in yeast, but their precise mode of molecular action remains to be identified. In contrast to the highly conserved subunits of the V-ATPase, orthologs of the yeast assembly factors are not easily identified based on sequence similarity. We show in this study that two ER-localized Arabidopsis proteins that share only 25% sequence identity with Vma21p can functionally replace this yeast assembly factor. Loss of AtVMA21a function in RNA interference seedlings caused impaired cell expansion and changes in Golgi morphology characteristic for plants with reduced V-ATPase activity, and we therefore conclude that AtVMA21a is the first V-ATPase assembly factor identified in a multicellular eukaryote. Moreover, VMA21p acts as a dedicated ER escort chaperone, a class of substrate-specific accessory proteins so far not identified in higher plants.

Background: The V-ATPase (VHA) is a protein complex of 13 different VHA-subunits. It functions as an ATP driven rotary-motor that electrogenically translocates H+ into endomembrane compartments. In Arabidopsis thaliana V-ATPase is encoded by 23 genes posing the question of specific versus redundant function of multigene encoded isoforms., Results: The transmembrane topology and stoichiometry of the proteolipid VHA-c" as well as the stoichiometry of the membrane integral subunit VHA-e within the V-ATPase complex were investigated by in vivo fluorescence resonance energy transfer (FRET). VHA-c", VHA-e1 and VHA-e2, VHA-a, VHA-c3, truncated variants of VHA-c3 and a chimeric VHA-c/VHA-c" hybrid were fused to cyan (CFP) and yellow fluorescent protein (YFP), respectively. The constructs were employed for transfection experiments with Arabidopsis thaliana mesophyll protoplasts. Subcellular localization and FRET analysis by confocal laser scanning microscopy (CLSM) demonstrated that (i.) the N- and C-termini of VHA-c" are localised in the vacuolar lumen, (ii.) one copy of VHA-c" is present within the VHA-complex, and (iii.) VHA-c" is localised at the ER and associated Golgi bodies. (iv.) A similar localisation was observed for VHA-e2, whereas (v.) the subcellular localisation of VHA-e1 indicated the trans Golgi network (TGN)-specifity of this subunit., Conclusion: The plant proteolipid ring is a highly flexible protein subcomplex, tolerating the incorporation of truncated and hybrid proteolipid subunits, respectively. Whereas the membrane integral subunit VHA-e is present in two copies within the complex, the proteolipid subunit VHA-c" takes part in complex formation with only one copy. However, neither VHA-c" isoform 1 nor any of the two VHA-e isoforms were identified at the tonoplast. This suggest a function in endomembrane specific VHA-assembly or targeting rather than proton transport.

Background: DNA-directed RNA polymerase II subunit F (POLR2F), a subunit of the V0 domain of the vacuolar ATPase (ATP6V0A1) and the prion protein (PRNP) are molecules of potential importance in carcinogenesis and targeted cancer therapy. However, their expression has not been studied in colorectal carcinomas., Patients and Methods: Expression microarray data were analyzed using a novel computational tool to reveal elevated levels of POLR2F, ATP6V0A1 and PRNP in relapsed colorectal carcinoma patients. The mRNA levels of POLR2F, ATP6V0A1 and PRNP were evaluated by quantitative RT-PCR in 70 colorectal carcinomas and 17 normal tissue specimens and were correlated with clinicopathological parameters., Results: POLR2F and PRNP were up-regulated in colorectal carcinomas. Moreover, a significant difference in the expression levels of all three molecules between the right colon and the rectum was observed. High expression levels of POLR2F and ATP6V0A1 correlated with improved 3-year survival. Moreover, PRNP expression constituted an independent prognostic factor of the 3-year survival in multivariate analysis., Conclusion: POLR2F and PRNP exhibited elevated levels in carcinomas compared to normal tissue samples suggesting a possible role for these molecules in colorectal cancer. The association of the three molecules with survival or disease prognosis warrants further investigation.

Mice deficient in the ATP6V1B1 ("B1") subunit of the vacuolar proton-pumping ATPase (V-ATPase) maintain body acid-base homeostasis under normal conditions, but not when exposed to an acid load. Here, compensatory mechanisms involving the alternate ATP6V1B2 ("B2") isoform were examined to explain the persistence of baseline pH regulation in these animals. By immunocytochemistry, the mean pixel intensity of apical B2 immunostaining in medullary A intercalated cells (A-ICs) was twofold greater in B1-/- mice than in B1+/+ animals, and B2 was colocalized with other V-ATPase subunits. No significant upregulation of B2 mRNA or protein expression was detected in B1-/- mice compared with wild-type controls. We conclude that increased apical B2 staining is due to relocalization of B2-containing V-ATPase complexes from the cytosol to the plasma membrane. Recycling of B2-containing holoenzymes between these domains was confirmed by the intracellular accumulation of B1-deficient V-ATPases in response to the microtubule-disrupting drug colchicine. V-ATPase membrane expression is further supported by the presence of "rod-shaped" intramembranous particles seen by freeze fracture microscopy in apical membranes of normal and B1-deficient A-ICs. Intracellular pH recovery assays show that significant (28-40% of normal) V-ATPase function is preserved in medullary ICs from B1-/- mice. We conclude that the activity of apical B2-containing V-ATPase holoenzymes in A-ICs is sufficient to maintain baseline acid-base homeostasis in B1-deficient mice. However, our results show no increase in cell surface V-ATPase activity in response to metabolic acidosis in ICs from these animals, consistent with their inability to appropriately acidify their urine under these conditions.

The RAVE complex is required for stable assembly of the yeast vacuolar proton-translocating ATPase (V-ATPase) during both biosynthesis of the enzyme and regulated reassembly of disassembled V(1) and V(0) sectors. It is not yet known how RAVE effects V-ATPase assembly. Previous work has shown that V(1) peripheral or stator stalk subunits E and G are critical for binding of RAVE to cytosolic V(1) complexes, suggesting that RAVE may play a role in docking of the V(1) peripheral stalk to the V(0) complex at the membrane. Here we provide evidence for an interaction between the RAVE complex and V(1) subunit C, another subunit that has been assigned to the peripheral stalk. The C subunit is unique in that it is released from both V(1) and V(0) sectors during disassembly, suggesting that subunit C may control the regulated assembly of the V-ATPase. Mutants lacking subunit C have assembly phenotypes resembling that of RAVE mutants. Both are able to assemble V(1)/V(0) complexes in vivo, but these complexes are highly unstable in vitro, and V-ATPase activity is extremely low. We show that in the absence of the RAVE complex, subunit C is not able to stably assemble with the vacuolar ATPase. Our data support a model where RAVE, through its interaction with subunit C, is facilitating V(1) peripheral stalk subunit interactions with V(0) during V-ATPase assembly.

Vacuolar-type proton ATPase (V-ATPase) is a multi-subunit enzyme that couples ATP hydrolysis to the translocation of protons across membranes. Mammalian cells express four isoforms of the a subunit of V-ATPase. Previously, we have shown that V-ATPase with the a3 isoform is highly expressed in pancreatic islets and is located in the membranes of insulin-containing granules in the beta cells. The a3 isoform functions in the regulation of hormone secretion. In this study, we have examined the distribution of a subunit isoforms in endocrine tissues, including the adrenal, parathyroid, thyroid, and pituitary glands, with isoform-specific antibodies. We have found that the a3 isoform is strongly expressed in all these endocrine tissues. Our results suggest that functions of the a3 isoform are commonly involved in the process of exocytosis in regulated secretion.

Adipose tissue synthesizes all components of the renin-angiotensin system. The renin receptor (RenR) is able, on renin binding, to increase its efficiency to generate angiotensin I from angiotensinogen. We demonstrate that RenR is specifically synthesized in the stromal portion of human adipose tissue in both isolated interadipocyte stromal cells and in stromal areas. RenR is expressed at the periphery of cells, strongly suggesting a membranal localization. RenR protein expression in primary cultures of human stromal cells decreased significantly during differentiation, whereas RenR mRNA levels did not change, demonstrating that RenR was expressed in both preadipocyte and nonpreadipocyte cells, and was regulated at a posttranscriptional level. Double-labeling immunohistochemistry of human adipose tissue sections revealed that RenR was colocalized with renin, whereas incubation of 3T3-L1, a preadipocyte cell line, with renin stimulated the phosphorylation state of the intracellular signaling pathway ERK 1/2, and short exposure of human adipose stromal cells in primary culture to renin was followed by a long-lasting dose-dependent increase of angiotensin I generation, indicating that adipose RenR is functional. We show, using a large set of human adipose tissue biopsies, that RenR expression was increased in visceral compared with subcutaneous adipose tissue of lean and obese patients. Taken together with our finding that RenR was colocalized with plasminogen activator inhibitor type 1, the main inhibitor of the fibrinolytic system in visceral adipose tissue, the above-mentioned data suggest that RenR plays a role in obesity-induced visceral adipose tissue accumulation and its accompanying cardiovascular complications.

Unlabelled: The Tcirg1 gene encodes the osteoclast-specific a3 isoform of the V-ATPase a subunit. Using the mouse osteoclastic model RAW264.7 cells, we studied Tcirg1 gene expression, and we identified PARP-1 as a transcriptional repressor negatively regulated by RANKL during osteoclastogenesis., Introduction: The TCIRG1 gene encodes the a3 isoform of the V-ATPase a subunit, and mutations at this locus account for approximately 60% of infantile malignant osteopetrosis cases. Using RAW264.7 cells as an osteoclastic differentiation model, we undertook a transcriptional study of the mouse Tcirg1 gene focused on the 4-kb region upstream of the transcription starting point., Materials and Methods: The promoter activity of serial-deletion fragments of the Tcirg1 gene promoter was monitored throughout the RAW264.7 cell differentiation process. We next performed EMSA, UV cross-linking, affinity purification, mass spectrometry analysis, gel supershift, and siRNA transfection experiments to identify the factor(s) interacting with the promoter., Results: The -3946/+113 region of the mouse Tcirg1 gene displayed a high basal promoter activity, which was enhanced by RANKL treatment of RAW264.7 cells. Constructs deleted up to -1589 retained this response to RANKL. A deletion up to -1402 induced a 3-fold enhancement of the basal activity, whereas RANKL response was not affected. EMSA experiments led us to identify within the -1589/-1402 region, a 10-nucleotide sequence, which bound a nuclear protein present in nondifferentiated RAW264.7 cells. This interaction was lost using nuclear extracts derived from RANKL-treated cells. Affinity purification followed by mass spectrometry analysis and gel supershift assay allowed the identification of poly(ADP-ribose) polymerase-1 (PARP-1) as this transcriptional repressor, whereas Western blot experiments revealed the cleavage of the DNA-binding domain of PARP-1 on RANKL treatment. Finally, both PARP-1 depletion after siRNA transfection and RAW264.7 cell treatment by an inhibitor of PARP-1 activity induced an increase of a3 mRNA expression., Conclusions: We provide evidence that the basal transcription activity of the Tcirg1 gene is negatively regulated by the binding of PARP-1 protein to its promoter region in mouse pre-osteoclast. On RANKL treatment, PARP-1 protein is cleaved and loses its repression effect, allowing an increase of Tcirg1 gene expression that is critical for osteoclast function.

Vacuolar proton ATPase accumulates protons inside various intracellular organelles such as synaptic vesicles; its membrane domain V0 could also be involved in membrane fusion. These different functions could require vacuolar proton ATPases possessing different V0 subunit a isoforms. In vertebrates, four genes encode isoforms a1-a4, and a1 variants are also generated by alternative splicing. We identified a novel a1 splice variant a1-IV and showed that the two a1 variants containing exon C are specifically expressed in neurons. Single neurons coexpress a2, a1-I, and a1-IV, and these subunit a isoforms are targeted to different membrane compartments. Recombinant a2 was accumulated in the trans-Golgi network, and a1-I was concentrated in axonal varicosities, whereas a1-IV was sorted to both distal dendrites and axons. Our results indicate that alternative splicing of exon N controls differential sorting of a1 variants to nerve terminals or distal dendrites, whereas exon C regulates their neuronal expression.

The cholangiocytes lining the intrahepatic bile ducts modify the primary secretion from the hepatocytes. The cholangiocytes secrete HCO (3)(-) into bile when stimulated with secretin in many species, including man. However, in rats, secretin stimulation neither affects biliary HCO (3)(-) concentration nor bile flow, whereas following bile duct ligation (BDL) it induces hypercholeresis with significant increase of NaHCO(3) concentration. We hypothesized that BDL might affect the expression of cholangiocyte H(+) transporters and thereby choleresis, and determined the expression and localization of the 31 kDa vacuolar type H(+)-ATPase (V-ATPase) subunit and of Na(+)/H(+) exchanger NHE3 in the livers of control and BDL rats by real-time PCR, in situ hybridization, immunoblotting, and immunohistochemistry. In controls, secretin had no effect on bile flow, whereas following BDL, secretin increased bile flow approximately threefold. V-ATPase and NHE3 were expressed in control cholangiocytes showing intracellular and apical distribution, respectively. BDL significantly up-regulated V-ATPase mRNA and protein expression and was associated with redistribution to the apical pole in approximately 60% of the cholangiocytes lining the small bile ductules. In contrast, NHE3 expression was significantly down-regulated by BDL at the mRNA and protein level. The data demonstrate expression of V-ATPase in rat cholangiocytes. BDL-induced down-regulation of NHE3 may contribute to a reduction of Na(+) and HCO (3)(-) reabsorption and thus to their net secretion into bile. Apical localization of V-ATPase in cholangiocytes may indicate its involvement in pH regulation and/or HCO (3)(-) salvage to compensate for NHE3 down-regulation in BDL.