Distal renal tubular acidosis as presenting manifestation of Wilson disease in a 11-year-old girl.

A 11-year-old girl was referred to the pediatric nephrology services of our hospital for evaluation of vitamin-D-refractory rickets. She was born to second-degree consanguineous parents. On examination, she had wrist widening and bilateral genu varum. She had normal anion gap metabolic acidosis, hypokalemia, and hyperchloremia. The fractional excretion of bicarbonate was 3% and the urine anion gap was positive. She also had hypercalciuria, but no phosphaturia, glucosuria or aminoaciduria. In view of a family history of an elder sister having rigidity with cognitive and speech impairment, an ophthalmic evaluation by slit lamp examination was performed in the index case that revealed bilateral Kayser-Fleischer rings. Serum ceruloplasmin was low and 24-h urine copper was elevated in the index case. Whole exome sequencing unveiled a novel pathogenic variant in exon 2 of the ATP7B gene (chr13: c.470del; Depth: 142x) (homozygous) that resulted in a frameshift and premature truncation of the protein, 15 amino acids downstream to codon 157 (p. Cys157LeufsTer15; NM_000053.4) confirming Wilson disease. There were no mutations in the ATP6V0A4, ATP6V1B1, SLC4A1, FOXI1, WDR72 genes or other genes that are known to cause distal RTA. Therapy with D-penicillamine and zinc supplements was initiated. A low dose of 2.5 mEq/kg/day of potassium citrate supplementation normalized the serum bicarbonate levels. This case was notable for the absence of hepatic or neurological involvement at admission. Wilson disease is well known to cause proximal renal tubular acidosis and Fanconi syndrome, with relatively lesser involvement of the distal renal tubules in the literature. However, isolated distal renal tubular involvement as presenting manifestation of Wilson disease (without hepatic or neurological involvement) is rare and can lead to diagnostic confusion.

The exceptional form and function of the giant bacterium Ca. Epulopiscium viviparus revolves around its sodium motive force.

Epulopiscium spp. are the largest known heterotrophic bacteria; a large cigar-shaped individual is a million times the volume of Escherichia coli. To better understand the metabolic potential and relationship of Epulopiscium sp. type B with its host Naso tonganus, we generated a high-quality draft genome from a population of cells taken from a single fish. We propose the name Candidatus Epulopiscium viviparus to describe populations of this best-characterized Epulopiscium species. Metabolic reconstruction reveals more than 5% of the genome codes for carbohydrate active enzymes, which likely degrade recalcitrant host-diet algal polysaccharides into substrates that may be fermented to acetate, the most abundant short-chain fatty acid in the intestinal tract. Moreover, transcriptome analyses and the concentration of sodium ions in the host intestinal tract suggest that the use of a sodium motive force (SMF) to drive ATP synthesis and flagellar rotation is integral to symbiont metabolism and cellular biology. In natural populations, genes encoding both F-type and V-type ATPases and SMF generation via oxaloacetate decarboxylation are among the most highly expressed, suggesting that ATPases synthesize ATP and balance ion concentrations across the cell membrane. High expression of these and other integral membrane proteins may allow for the growth of its extensive intracellular membrane system. Further, complementary metabolism between microbe and host is implied with the potential provision of nitrogen and B vitamins to reinforce this nutritional symbiosis. The few features shared by all bacterial behemoths include extreme polyploidy, polyphosphate synthesis, and thus far, they have all resisted cultivation in the lab.

Ketone Body Exposure of Cardiomyocytes Impairs Insulin Sensitivity and Contractile Function through Vacuolar-Type H+-ATPase Disassembly-Rescue by Specific Amino Acid Supplementation.

The heart is metabolically flexible. Under physiological conditions, it mainly uses lipids and glucose as energy substrates. In uncontrolled diabetes, the heart switches towards predominant lipid utilization, which over time is detrimental to cardiac function. Additionally, diabetes is accompanied by high plasma ketone levels and increased utilization of energy provision. The administration of exogenous ketones is currently being investigated for the treatment of cardiovascular disease. Yet, it remains unclear whether increased cardiac ketone utilization is beneficial or detrimental to cardiac functioning. The mechanism of lipid-induced cardiac dysfunction includes disassembly of the endosomal proton pump (named vacuolar-type H+-ATPase; v-ATPase) as the main early onset event, followed by endosomal de-acidification/dysfunction. The de-acidified endosomes can no longer serve as a storage compartment for lipid transporter CD36, which then translocates to the sarcolemma to induce lipid accumulation, insulin resistance, and contractile dysfunction. Lipid-induced v-ATPase disassembly is counteracted by the supply of specific amino acids. Here, we tested the effect of ketone bodies on v-ATPase assembly status and regulation of lipid uptake in rodent/human cardiomyocytes. 3-ß-hydroxybutyrate (3HB) exposure induced v-ATPase disassembly and the entire cascade of events leading to contractile dysfunction and insulin resistance, similar to conditions of lipid oversupply. Acetoacetate addition did not induce v-ATPase dysfunction. The negative effects of 3HB could be prevented by addition of specific amino acids. Hence, in sedentary/prediabetic subjects ketone bodies should be used with caution because of possible aggravation of cardiac insulin resistance and further loss of cardiac function. When these latter maladaptive conditions would occur, specific amino acids could potentially be a treatment option.

Synthesis, Cytotoxicity, Anti-Migration and Anti-Invasion Activity of Diphyllin Heterocyclic Derivatives.

BACKGROUND: Diphyllin, an arylnaphthalene lignan lactone, isolated from many traditional medicinal plants, has been reported to possess anticancer and antiviral activities. Natural diphyllin and its glycosides were identified as potent vacuolar H+-ATPase (V-ATPase) inhibitors. OBJECTIVE: The aim of this study was to design and synthesize a series of heterocyclic derivatives of diphyllin as novel anticancer agents. METHODS: The targeted heterocyclic derivatives of diphyllin were synthesized from diphyllin employing etherification reaction and N-substitution reaction. Cytotoxicity of these compounds on four cancer cells was assessed by MTT assay. The inhibitory activity of V-ATPase of compound 3n was measured on MGC-803 cells. Anti-migration and anti-invasion abilities were assessed by transwell invasion assay and scratch wound assay. RESULTS: Most of these derivatives displayed potent cytotoxicity on four cancer cells at submicromolar concentrations. The most potent derivative 3n has been shown to inhibit V-ATPase activity, migration and invasion abilities on MGC-803 cells at 0.75 µM. CONCLUSION: The collective results clearly indicate that heterocyclic derivatives of diphyllin inhibit the viability, V-ATPase activity, migration and invasion of the MGC803 cells. The current findings provide valuable insights for the future development of novel diphyllin derivatives as anticancer agents.

Specific amino acid supplementation rescues the heart from lipid overload-induced insulin resistance and contractile dysfunction by targeting the endosomal mTOR-v-ATPase axis.

OBJECTIVE: The diabetic heart is characterized by extensive lipid accumulation which often leads to cardiac contractile dysfunction. The underlying mechanism involves a pivotal role for vacuolar-type H+-ATPase (v-ATPase, functioning as endosomal/lysosomal proton pump). Specifically, lipid oversupply to the heart causes disassembly of v-ATPase and endosomal deacidification. Endosomes are storage compartments for lipid transporter CD36. However, upon endosomal deacidification, CD36 is expelled to translocate to the sarcolemma, thereby inducing myocardial lipid accumulation, insulin resistance, and contractile dysfunction. Hence, the v-ATPase assembly may be a suitable target for ameliorating diabetic cardiomyopathy. Another function of v-ATPase involves the binding of anabolic master-regulator mTORC1 to endosomes, a prerequisite for the activation of mTORC1 by amino acids (AAs). We examined whether the relationship between v-ATPase and mTORC1 also operates reciprocally; specifically, whether AA induces v-ATPase reassembly in a mTORC1-dependent manner to prevent excess lipids from entering and damaging the heart. METHODS: Lipid overexposed rodent/human cardiomyocytes and high-fat diet-fed rats were treated with a specific cocktail of AAs (lysine/leucine/arginine). Then, v-ATPase assembly status/activity, cell surface CD36 content, myocellular lipid uptake/accumulation, insulin sensitivity, and contractile function were measured. To elucidate underlying mechanisms, specific gene knockdown was employed, followed by subcellular fractionation, and coimmunoprecipitation. RESULTS: In lipid-overexposed cardiomyocytes, lysine/leucine/arginine reinternalized CD36 to the endosomes, prevented/reversed lipid accumulation, preserved/restored insulin sensitivity, and contractile function. These beneficial AA actions required the mTORC1-v-ATPase axis, adaptor protein Ragulator, and endosomal/lysosomal AA transporter SLC38A9, indicating an endosome-centric inside-out AA sensing mechanism. In high-fat diet-fed rats, lysine/leucine/arginine had similar beneficial actions at the myocellular level as in vitro in lipid-overexposed cardiomyocytes and partially reversed cardiac hypertrophy. CONCLUSION: Specific AAs acting through v-ATPase reassembly reduce cardiac lipid uptake raising the possibility for treatment in situations of lipid overload and associated insulin resistance.

Cimetidine-induced androgenic failure causes cell death and changes in actin, EGF and V-ATPase immunoexpression in rat submandibular glands.

Submandibular gland (SMG) is responsive to androgens via androgen receptor (AR). We verified whether cimetidine induces androgenic dysfunction in SMG, and evaluated the structural integrity, cell death and immunoexpression of actin, EGF and V-ATPase in androgen-deficient SMG. Male rats received cimetidine (CMTG) and control animals (CG) received saline. Granular convoluted tubules (GCTs) diameter and number of acinar cell nuclei were evaluated. TUNEL and immunofluorescence reactions for detection of AR, testosterone, actin, EGF and V-ATPase were quantitatively analysed. In CG, testosterone immunolabelling was detected in acinar and ductal cells cytoplasm. AR-immunolabelled nuclei were observed in acinar cells whereas ductal cells showed AR-immunostained cytoplasm, indicating a non-genomic AR action. In CMTG, the weak testosterone and AR immunoexpression confirmed cimetidine-induced androgenic failure. A high cell death index was correlated with decreased number of acinar cells, GCTs diameter and EGF immunoexpression under androgenic dysfunction. Actin immunofluorescence decreased in the SMG cells, but an increased and diffuse cytoplasmic V-ATPase immunolabelling was observed in striated ducts, suggesting a disruption in the actin-dependent V-ATPase recycling due to androgenic failure. Our findings reinforce the androgenic role in the maintenance of SMG histophysiology, and point to a potential clinical use of cimetidine against androgen-dependent glandular tumour cells.

Ammonium Accumulation Caused by Reduced Tonoplast V-ATPase Activity in Arabidopsis thaliana.

Plant vacuoles are unique compartments that play a critical role in plant growth and development. The vacuolar H+-ATPase (V-ATPase), together with the vacuolar H+-pyrophosphatase (V-PPase), generates the proton motive force that regulates multiple cell functions and impacts all aspects of plant life. We investigated the effect of V-ATPase activity in the vacuole on plant growth and development. We used an Arabidopsisthaliana (L.) Heynh. double mutant, vha-a2 vha-a3, which lacks two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a. The mutant is viable but exhibits impaired growth and leaf chlorosis. Nitrate assimilation led to excessive ammonium accumulation in the shoot and lower nitrogen uptake, which exacerbated growth retardation of vha-a2 vha-a3. Ion homeostasis was disturbed in plants with missing VHA-a2 and VHA-a3 genes, which might be related to limited growth. The reduced growth and excessive ammonium accumulation of the double mutant was alleviated by potassium supplementation. Our results demonstrate that plants lacking the two tonoplast-localized subunits of V-ATPase can be viable, although with defective growth caused by multiple factors, which can be alleviated by adding potassium. This study provided a new insight into the relationship between V-ATPase, growth, and ammonium accumulation, and revealed the role of potassium in mitigating ammonium toxicity.

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.

Abeliophyllum distichum Nakai alleviates postmenopausal osteoporosis in ovariectomized rats and prevents RANKL-induced osteoclastogenesis in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Abeliophyllum distichum Nakai (AD), called Miseon, is one of Korea's monotypic endemic species. As a folk remedy, the AD has been used to treat inflammatory disease, stomachaches, diarrhea, and gynecologic disease in Korea. Some researchers have reported that the AD has anti-cancer, anti-inflammatory, and anti-oxidant effects. But the protective effect of AD leaf for osteoporosis has not been reported yet. AIM OF THE STUDY: This study aimed to analyze the effects and mechanism of AD-ethyl acetate fraction (EA) extract on the osteoporosis, one of the gynecologic disease. MATERIALS AND METHODS: The RAW 264.7 cells were used as a model for RANKL-induced osteoclastogenesis. We measured the TRAcP activity, expressions of NFATc1, c-fos, and MAPK to investigate the effect of AD-EA. OVX-induced osteoporosis rat model was used as menopausal osteoporosis. After both ovaries were removed through a surgical procedure, and AD-EA or 17b-estradiol was orally administered for 8 weeks. BMD of femurs was measured as well as the bone morphometric parameter, such as BV/TV, trabecular thickness, number and surface using a micro CT. RESULTS: AD-EA significantly inhibited TRAcP activity, actin ring formation, pit formation and the expressions of osteoclast-related genes in a dose-dependent manner through the inhibition of the MAPK and c-fos/NFATc1 pathway. In addition, low dose administration of AD-EA improved the deterioration of trabecular bone microarchitecture caused by OVX through the inhibition of bone resorption by TRAcP and CTK. CONCLUSIONS: These results suggest that AD-EA may contribute to the therapy of osteoporosis caused by menopause in women.

Cysteine Toxicity Drives Age-Related Mitochondrial Decline by Altering Iron Homeostasis.

Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease. Despite the long-standing connection between these organelles, the function(s) of lysosomes required to sustain mitochondrial health remains unclear. Here, working in yeast, we show that the lysosome-like vacuole maintains mitochondrial respiration by spatially compartmentalizing amino acids. Defects in vacuole function result in a breakdown in intracellular amino acid homeostasis, which drives age-related mitochondrial decline. Among amino acids, we find that cysteine is most toxic for mitochondria and show that elevated non-vacuolar cysteine impairs mitochondrial respiration by limiting intracellular iron availability through an oxidant-based mechanism. Cysteine depletion or iron supplementation restores mitochondrial health in vacuole-impaired cells and prevents mitochondrial decline during aging. These results demonstrate that cysteine toxicity is a major driver of age-related mitochondrial deterioration and identify vacuolar amino acid compartmentation as a cellular strategy to minimize amino acid toxicity.

Impaired lysosomal acidification triggers iron deficiency and inflammation in vivo.

Lysosomal acidification is a key feature of healthy cells. Inability to maintain lysosomal acidic pH is associated with aging and neurodegenerative diseases. However, the mechanisms elicited by impaired lysosomal acidification remain poorly understood. We show here that inhibition of lysosomal acidification triggers cellular iron deficiency, which results in impaired mitochondrial function and non-apoptotic cell death. These effects are recovered by supplying iron via a lysosome-independent pathway. Notably, iron deficiency is sufficient to trigger inflammatory signaling in cultured primary neurons. Using a mouse model of impaired lysosomal acidification, we observed a robust iron deficiency response in the brain, verified by in vivo magnetic resonance imaging. Furthermore, the brains of these mice present a pervasive inflammatory signature associated with instability of mitochondrial DNA (mtDNA), both corrected by supplementation of the mice diet with iron. Our results highlight a novel mechanism linking impaired lysosomal acidification, mitochondrial malfunction and inflammation in vivo.

Cleistanthus collinus poisoning affects mitochondrial respiration and induces oxidative stress in the rat kidney.

Cleistanthus collinus is a poisonous shrub used for deliberate self-harm in rural areas of South India and intake of boiled decoction of leaves is a common method of self-harm. Distal renal tubular acidosis (dRTA) is an important clinical symptom observed in C. collinus poisoning, and renal V-ATPases may be potential targets of damage. However, a lack of understanding of molecular mediators involved hampers medical management, which is mainly supportive. We hypothesized that C. collinus poisoning induces renal oxidative stress; probably by inducing mitochondrial uncoupling, which compromises V-ATPase activity to ultimately produce dRTA. This was tested by exposing renal BBMV, kidney cells in culture, and Wistar rats to C. collinus poisoning. Exposure to C. collinus aqueous extract resulted in significant elevations in the lipid peroxidation marker, conjugated dienes, in cell culture and in vivo. A significant decrease in mitochondrial respiratory control ratio was observed in kidneys from C. collinus-treated animals suggesting that mitochondrial oxidative phosphorylation is uncoupled. This was accompanied by significant increase in ADP levels and a decrease in proton pump activity. Thus, these results demonstrate that C. collinus poisoning induces oxidative stress which influences proton pump activity, probably due to feedback inhibition by elevated ADP levels because of mitochondrial dysfunction in the rat kidney.

Curcumin Treatment in Combination with Glucose Restriction Inhibits Intracellular Alkalinization and Tumor Growth in Hepatoma Cells.

Dysregulation of cellular energy metabolism is closely linked to cancer development and progression. Calorie or glucose restriction (CR or GR) inhibits energy-dependent pathways, including IGF-1/PI3K/Akt/mTOR, in cancer cells. However, alterations in proton dynamics and reversal of the pH gradient across the cell membrane, which results in intracellular alkalinization and extracellular acidification in cancer tissues, have emerged as important etiopathogenic factors. We measured glucose, lactate, and ATP production after GR, plant-derived CR-mimetic curcumin treatment, and curcumin plus GR in human hepatoma cells. Intracellular pH regulatory effects, in particular, protein-protein interactions within mTOR complex-1 and its structural change, were investigated. Curcumin treatment or GR mildly inhibited Na+/H+ exchanger-1 (NHE1). vATPase, monocarboxylate transporter (MCT)-1, and MCT4 level. Combination treatment with curcumin and GR further enhanced the inhibitory effects on these transporters and proton-extruding enzymes, with intracellular pH reduction. ATP and lactate production decreased according to pH change. Modeling of mTOR protein revealed structural changes upon treatments, and curcumin plus GR decreased binding of Raptor and GßL to mTOR, as well as of Rag A and Rag B to Raptor. Consequently, 4EBP1 phosphorylation was decreased and cell migration and proliferation were inhibited in a pH-dependent manner. Autophagy was increased by curcumin plus GR. In conclusion, curcumin treatment combined with GR may be a useful supportive approach for preventing intracellular alkalinization and cancer progression.

Effects of Marantodes pumilum (Kacip Fatimah) on vaginal pH and expression of vacoular ATPase and carbonic anhydrase in the vagina of sex-steroid deficient female rats.

BACKGROUND: In sex-steroid deficiency, increased in the pH of vaginal fluid is due to low estrogen levels. HYPOTHESIS: Consumption of Marantodes pumilum leaves helps to ameliorate increased in vaginal fluid pH in sex-steroid deficient condition. PURPOSE: To investigate changes in vaginal fluid pH and expression of proteins that participate in pH changes i.e vacoular (V)-ATPases and carbonic anhydrases (CA) in the vagina following M. pumilum leaves consumption. METHODS: Ovariectomized adult female rats were treated orally with M. pumilum leaves extract (MPE) at 100, 250 and 500 mg/kg.b.w and estradiol at 0.2 µg/kg/b.w for 28 days. At the end of the treatment, vaginal fluid pH was measured in anesthetised rats by using micropH probe. Following sacrificed, levels of V-ATPase and CA proteins and mRNAs in the vagina were identified by Western blotting and real-time PCR, respectively. Protein distribution was visualized by immunohistochemistry. RESULTS: Administration of MPE causes the pH of vaginal fluid to decrease and expression and distribution of vaginal V-ATPase A & B and CA II, III, IX, XII and XIII to increase. CONCLUSIONS: The decrease in vaginal fluid pH following MPE treatment suggested that this herb has potential to be used to ameliorate vaginal fluid pH changes in sex-steroid deficient condition.

Characterization of the Genes Involved in Malic Acid Metabolism from Pear Fruit and Their Expression Profile after Postharvest 1-MCP/Ethrel Treatment.

In this study, five genes involved in malic acid (MA) metabolism, including a cytosolic NAD-dependent malate dehydrogenase gene ( cyNAD-MDH), a cytosolic NADP-dependent malic enzyme gene ( cyNADP-ME), two vacuolar H+-ATPase genes ( vVAtp1 and vVAtp2), and one vacuolar inorganic pyrophosphatase gene ( vVPp), were characterized from pear fruit based on bioinformatic and experimental analysis. Their expression profile in "Housui" pear was tissue-specific, and their expression patterns during fruit development were diverse. During "Housui" pear storage, MA content decreased, which was associated with the downregulated transcripts of MA metabolism-related genes and cyNAD-MDH activity and higher cyNADP-ME activity. The response of MA metabolism to postharvest 1.5 µL L-1 1-MCP fumigation and 0.5 mL L-1 ethrel dipping was distinct: 1-MCP fumigation upregulated gene expression and cyNAD-MDH activity and suppressed cyNADP-ME activity, and thus maintained higher MA abundance when compared with those in the control; on the other hand, an opposite behavior was observed in ethrel-treated fruit.

Isolation of Vacuoles from the Leaves of the Medicinal Plant Catharanthus roseus.

The isolation of vacuoles is an essential step to unravel the important and complex functions of this organelle in plant physiology. Here, we describe a method for the isolation of vacuoles from Catharanthus roseus leaves involving a simple procedure for the isolation of protoplasts, and the application of a controlled osmotic/thermal shock to the naked cells, leading to the release of intact vacuoles, which are subsequently purified by density gradient centrifugation. The purity of the isolated intact vacuoles is assayed by microscopy, western blotting, and measurement of vacuolar (V)-H+-ATPase hydrolytic activity. Finally, membrane functionality and integrity is evaluated by measuring the generation of a transtonoplast pH gradient by the V-H+-ATPase and the V-H+-pyrophosphatase, also producing further information on vacuole purity.

Poligoni Multiflori Radix enhances osteoblast formation and reduces osteoclast differentiation.

Poligoni Multiflori Radix (PMR) is a traditional Korean medicinal herb that is known to have various pharmacological effects, including antihyperlipidemic, anticancer, and anti­inflammatory effects. However, the effects of PMR on bone metabolism have not been elucidated to date. The present study aimed to investigate the in vitro and in vivo effect of PMR water extract on the regulation of osteoblast and osteoclast activity. Effects of PMR water extract on receptor activator of nuclear factor­kB ligand (RANKL)­induced osteoclast differentiation and survival of mouse bone marrow macrophages (BMMs) obtained from femurs were investigated by tartrate­acid resistant acid phosphatase (TRAP)­positive cells and XTT assay. Expression of osteoclast­related genes was assayed by western blot analysis and reverse transcription­quantitative polymerase chain reaction. Additionally, the effects of PMR water extract on osteoblastic proliferation and differentiation were investigated by alkaline phosphatase (ALP) activity assay, alizarin red staining, and levels of mRNA encoding known osteoblast markers. Furthermore, the effects of PMR water extract on lipopolysaccharide (LPS)­induced bone loss were examined in a mouse model. PMR inhibited RANKL­induced osteoclast differentiation of BMMs in a dose­dependent manner without significant cytotoxicity, and suppressed expression of the main osteoclast differentiation markers Fos proto­oncogene and nuclear factor of activated T­cell. In addition, PMR decreased the mRNA expression levels of NFATc1 target genes, including TRAP, osteoclast­associated receptor, ATPase H+ transporting, lysosomal 38 kDa V0 subunit d2, and Cathepsin K. These inhibitory effects were mediated by the p38 and extracellular signal­regulated kinase/nuclear factor­κB pathway. Simultaneously, PMR enhanced the differentiation of primary osteoblasts, and increased the mRNA expression of runt­related transcription factor 2, ALP, osterix, and osteocalcin. Notably, PMR improved LPS­induced trabecular bone loss in mice. Collectively, the present findings demonstrated that PMR may regulate bone remodeling by reducing osteoclast differentiation and stimulating osteoblast formation. These results suggest that PMR may be used for the treatment of bone diseases, such as osteoporosis and rheumatoid arthritis.

Misrouting of v-ATPase subunit V0a1 dysregulates lysosomal acidification in a neurodegenerative lysosomal storage disease model.

Defective lysosomal acidification contributes to virtually all lysosomal storage disorders (LSDs) and to common neurodegenerative diseases like Alzheimer's and Parkinson's. Despite its fundamental importance, the mechanism(s) underlying this defect remains unclear. The v-ATPase, a multisubunit protein complex composed of cytosolic V1-sector and lysosomal membrane-anchored V0-sector, regulates lysosomal acidification. Mutations in the CLN1 gene, encoding PPT1, cause a devastating neurodegenerative LSD, INCL. Here we report that in Cln1-/- mice, which mimic INCL, reduced v-ATPase activity correlates with elevated lysosomal pH. Moreover, v-ATPase subunit a1 of the V0 sector (V0a1) requires palmitoylation for interacting with adaptor protein-2 (AP-2) and AP-3, respectively, for trafficking to the lysosomal membrane. Notably, treatment of Cln1-/- mice with a thioesterase (Ppt1)-mimetic, NtBuHA, ameliorated this defect. Our findings reveal an unanticipated role of Cln1 in regulating lysosomal targeting of V0a1 and suggest that varying factors adversely affecting v-ATPase function dysregulate lysosomal acidification in other LSDs and common neurodegenerative diseases.

The vacuolar-ATPase complex and assembly factors, TMEM199 and CCDC115, control HIF1α prolyl hydroxylation by regulating cellular iron levels.

Hypoxia Inducible transcription Factors (HIFs) are principally regulated by the 2-oxoglutarate and Iron(II) prolyl hydroxylase (PHD) enzymes, which hydroxylate the HIFα subunit, facilitating its proteasome-mediated degradation. Observations that HIFα hydroxylation can be impaired even when oxygen is sufficient emphasise the importance of understanding the complex nature of PHD regulation. Here, we use an unbiased genome-wide genetic screen in near-haploid human cells to uncover cellular processes that regulate HIF1α. We identify that genetic disruption of the Vacuolar H+ ATPase (V-ATPase), the key proton pump for endo-lysosomal acidification, and two previously uncharacterised V-ATPase assembly factors, TMEM199 and CCDC115, stabilise HIF1α in aerobic conditions. Rather than preventing the lysosomal degradation of HIF1α, disrupting the V-ATPase results in intracellular iron depletion, thereby impairing PHD activity and leading to HIF activation. Iron supplementation directly restores PHD catalytic activity following V-ATPase inhibition, revealing important links between the V-ATPase, iron metabolism and HIFs.

Anti-Osteoclastic Activity of Artemisia capillaris Thunb. Extract Depends upon Attenuation of Osteoclast Differentiation and Bone Resorption-Associated Acidification Due to Chlorogenic Acid, Hyperoside, and Scoparone.

The present study attempts to elucidate the anti-osteoporotic activity of Artemisia capillaris Thunb. in the form of anti-osteoclastic effect and responsible bioactive compounds. The contents of chlorogenic acid, caffeic acid, hyperoside, isoquercitrin, isochlorogenic acid A, and scoparone in Artemisia capillaris hydroethanolic extract (ACHE) were 38.53, 0.52, 4.07, 3.03, 13.90, and 6.59 mg/g, respectively. ACHE diminished osteoclast differentiation and bone resorption due to chlorogenic acid, hyperoside, and scoparone. In addition, ACHE attenuated acidification as well as reducing tumor necrosis factor receptor-associated factor 6 (TRAF6) expression and its association with vacuolar H⁺-adenosine triphosphatase (V-ATPase). Furthermore, chlorogenic acid, hyperoside, and scoparone from A. capillaris abrogated the association of V-ATPase with TRAF6, suggesting that the blockage of bone resorption by A. capillaris was partially mediated by reducing acidification through down-regulating interaction of V-ATPase with TRAF6 due to scoparone as well as chlorogenic acid and hyperoside. These results imply that the anti-osteoclastic effect of A. capillaris through down-regulating osteoclast differentiation and bone resorption may contribute to its anti-osteoporotic effect.