To understand better how Ca2+ and H2O2 signals are integrated to enhance grass acclimation to stress conditions, we analyzed the rearrangements of endogenous ion accumulation, antioxidant systems and secondary metabolism in roots, stems and leaves of perennial ryegrass (Lolium perenne L.) treated with exogenous Ca2+ and H2O2 under salinity. Ca2+ signaling remarkably enhanced the physiological response to salt conditions. Ca2+ signaling could maintain ROS homeostasis in stressed grass by increasing the responses of antioxidant genes, proteins and enzymes. H2O2 signaling could activate ROS homeostasis by inducing antioxidant genes but weakened Ca2+ signaling in leaves. Furthermore, the metabolic profiles revealed that sugars and sugar alcohol accounted for 49.5–88.2% of all metabolites accumulation in all treated leaves and roots. However, the accumulation of these sugars and sugar alcohols displayed opposing trends between Ca2+ and H2O2 application in salt-stressed plants, which suggests that these metabolites are the common regulatory factor for Ca2+ and H2O2 signals. These findings assist in understanding better the integrated network in Ca2+ and H2O2 of cool-season turfgrass’ response to salinity.Little is known about the interplay between Ca Some studies demonstrated that Ca2+ could activate CAT enzyme activity to minimize cellular H2O2 level, and function as an upstream component in H2O2 stress signals in plantset al.2O2 may function as an upstream signal to regulate Ca2+ in NaCl-stressed Populus euphratica cells. Despite their relevance in many cell biology aspects, integrated changes in spatio-temporal regulation of H2O2 and Ca2+ signaling remains elusive in stressed plants710Calcium Ca+ and hyd2O2, HO and O2−, could be triggered by environmental stresses2− → H2O2 → HO → H2O is catalyzed by different antioxidant enzymes such as superoxide dismutase , peroxidase , ascorbate peroxidase , catalase 1314162O2, which catalyze the reduction of H2O2 to water in plant organelles2O2 in chloroplasts, mitochondria, peroxisomes and apoplastic space. CAT on the other hand acts in glyoxysomes and peroxisomes, which minimize cellular H2O2 level2O2 accumulation may increase CAT activity or CAT gene expression2O2 treatment could activate Ca2+ channels and simultaneously mediate Ca2+ influx in protoplasts and increase of [Ca2+]cyt in intact guard cells2+/CaM could down-regulate H2O2 levels in plants by stimulating the catalytic activity of plant CAT. Jiang and Huang2+ could increase CAT activity in heat stressed grasses. The regulatory mechanisms among Ca2+ and H2O2 signaling, and antioxidant enzymes also remain unclear, while several lines of evidence indicate one interaction between their multiple signal transduction.Reactive oxygen species (ROS) accumulation, such as H2+ and H2O2 signaling involves many genes and metabolites in stressed plant522et al.2+ treatment in bermudagrass [Cynodon dactylon (L.) Pers.] in response to cold stress. Dittami et al.2O2 treatment in brown alga Ectocarpus siliculosus. Furthermore, previous studies have demonstrated that the majority of the expressed genes could be activated by these metabolites, which may be the gene regulation end productset al.2+]cyt might initiate the expression of genes, such as AtSUC1 and AtSUC2, involved in biosynthesis of storage carbohydrate. On the contrary, exogenous application of hexoses also might mediate by monosaccharide-H+ co-transporters to increase [Ca2+]cyt level. In addition, H2O2 signaling activating gene expression also involved metabolites such as proline and sucrose2+ and H2O2 signaling may be regarded as a major regulation character in plants in response to stress environment.Many studies have revealed that regulation of CaLolium perenne L.) is an extensively utilized cool-season grass globally used for forage, turfgrass and soil stabilization in United States, Australia, New Zealand, Japan and most of European countries because of its rapid establishment rate122+ or H2O2 on the secondary metabolism and oxidative signaling in perennial ryegrass under salt stress. The objective of this study was to further understand the orchestrated response of antioxidant system and metabolisms induced by H2O2/Ca2+ signaling in cool-season turfgrass in response to salt stress.Salinity is a major abiotic stress and increasingly threatens agriculture and food production worldwide2O2, but minimized the turf quality score in perennial ryegrass leaves. However, 7 mM Ca(NO3)2·4H2O added in 300 mM NaCl treatment solution reduced the levels of MDA, EL and H2O2, and increased the turf quality score compared with 300 mM NaCl treated plants  and ROS metabolism genes  were analyzed in perennial ryegrass leaves and roots. When exposed to all treatments, the transcripts levels of APX remained down-regulated in leaves  and excellent resolution were resolved from each polar extract. Based on the internal consistency of RT and retention indices (RI), 41 metabolites could be identified across all samples in this study  BP, but greater content of talose, glucoheptose and allose (5TMS) MP in comparison with controls. In contrast, both NaCl and H2O2 chemical treatments stimulated higher abundance of tagatose, idose, sucrose, psicose and allose (5TMS) BP, but lower glucoheptose abundance compared with controls  BP, sucrose, cellobiose and inositol abundance had lower accumulation compared with NaCl stress alone, but eventually declined. In addition, plant roots accumulated lower tagatose, psicose, idose, allose (5TMS) BP, glucose, sucrose, cellobiose and inositol under NaCl treatment without Ca2+ compared with controls. However, the treatment after Ca2+ addition increased sucrose content  BP abundance, but lower glucose, sucrose and cellobiose accumulation. Idose, allose (5TMS) BP, sucrose showed greater abundance under both NaCl and H2O2 chemical treatments than H2O2 alone. In contrast, both NaCl and H2O2 chemical treatments induced lower cellobiose content than H2O2 alone  genes, such as CaM1 and CaM2, could regulate different target intermediate in Ca2+ mediated signal transduction2O2 stress induced CaM1 and CaM2 expression in leaves. However, the H2O2 stressed roots exhibited the same CaM1 transcript level as the controls. Consistently, the strong induction of CaM1 and CaM2 in NaCl stressed leaves and roots reinforces the recent suggestion that H2O2 may function as an upstream signal activating Ca2+ signals in NaCl stressed perennial ryegrassBoth Na2O2 is one of the major active oxygen species and could be synthesized by the catalyzed reaction of SOD2O2 level while SOD activity and CytCu/ZnSOD and MnSOD expression level increased in leaves of perennial ryegrass. In salt-stressed roots, the H2O2 level significantly increased with an increasing expression of ChlCu/ZnSOD, CytCu/ZnSOD and MnSOD. Excessive H2O2 could be detoxified into H2O by the catalyzed reaction of CAT, GPX, APX or GR in plants cellsCAT, GPX, APX or GRCAT and APX had higher transcript abundance than SOD genes such as ChlCu/ZnSOD, CytCu/ZnSOD and MnSOD in NaCl-stressed root. The same result was obtained under H2O2 treatment and H2O2+ NaCl treatment, suggesting that self-regulation for ROS homeostasis could be activated due to the excessive accumulation of H2O2 induced by NaCl stress. Together with exogenous Ca2+ signal could increase SOD, POD, CAT and APX activity, as well as may function as a downstream signal activated by H2O2, we speculated that Ca2+ and H2O2 signals may exist one common regulation orientation that maintains ROS homeostasis in salt-stressed cool-season turfgrass.H2+ and H2O2 signals affect metabolite patterns in salt-stressed cool-season turfgrass. Here, 41 metabolites exhibited different response patterns in plants exposed to NaCl, Ca2+ or H2O2. Consistent with the previous research in barley (Hordeum vulgare L.)Arabidopsis thaliana2+ addition during the first 4 days exposure. In contrast, removing Ca2+ or adding H2O2 accelerated the upward trend. At 8 DAT, upon Ca2+ addition treatment obtained the highest accumulation compared with other treatment, but H2O2 alone treatment had the lowest metabolic accumulation during the 8 days treatment. The present data combined with previous findings involved in metabolites response under salt stress, may help to understand better the molecular basis of Ca2+ and ROS signaling in salt-treated plants.Since the accumulation of various metabolites changed when plants were exposed to salinity conditions2+ and ROS signaling in salt-treated leaves. First, NaCl treatment induced the production of tagatose, idose, sucrose, talose, allose (5TMS) BP and psicose but inhibited glucose and inositol content in leaves. However, we found that all eleven sugars and one sugar alcohol showed higher accumulation after Ca2+ application compared with control or NaCl treatment lines in leaves. On the contrary, NaCl treatment induced higher sugar than that in Ca2+ application lines in root. These results suggest that up-regulated sugars induced by Ca2+ signaling in leaf could contribute to the stronger response to salt stress. Secondly, the lowest contribution rate (49.5%) for sugars in total metabolites content was just obtained at H2O2 treatment alone at 8 DAT. Correspondingly, in leaves, we found that H2O2 treatment alone induced lower accumulation of tagatose, idose, glucose, sucrose, cellobiose, psicose and allose (5TMS) BP compared with controls or NaCl treatment lines, and just the talose, glucoheptose and allose (5TMS) MP content increased, which indicated that the positive role induced by Ca2+ signaling in leaf may be inhibited by H2O2 signaling. However, converse results were observed in stressed roots, which may be in relation to the different carbohydrate allocation in source and sink tissue under salt stressSugars not only directly provide energy and solutes for osmotic adjustment452O2 and Ca2+-based signaling response through coupling ion accumulation, antioxidant systems and secondary metabolism analysis in cool-season turfgrass. Increased Ca2+/Mg2+ ratio but decreased Na+/K+ ratio were observed following external Ca2+ application in salt-stressed grass. Exogenous Ca2+ signaling could induce higher antioxidant enzymes activity and the expression level of antioxidant gene and protein in plants to adapt salinity environment. However, H2O2 application decreased Ca2+ signaling but increased the transcript abundance of CAT and APX. In order to maintain ROS homeostasis, Ca2+ and H2O2 signals had one common regulation pattern in salt-stressed cool-season turfgrass. In addition, H2O2/Ca2+-mediated metabolites detected in this study could provide a dataset of common regulatory factors for signaling transduction and salinity acclimation in perennial ryegrass. These findings involved in overlapping roles of H2O2/Ca2+ signaling in salinity response mechanisms supply one novel strategy for a cool-season turfgrass’s adaptation under salt stress.In conclusion, the present study reported here reinforces the understanding of salt tolerance involved in H−2s−1, and a 14 h photoperiod) throughout the experiment.Perennial ryegrass ‘Quick start II’ seeds were planted into plastic pots  with sand as substrate. After seedlings had grown to 8 cm tall, they were mowed to a height of 5 cm and were irrigated twice weekly with half-strength Hoagland’s solutionet al.2+ treatment line, were treated with 300 mM NaCl + 7 mM Ca(NO3)2·4H2O for 12 d. All treatment solutions were prepared in half-strength Hoagland’s solution. Shoots samples for physiological, gene expression and metabolic analysis were harvested at 0, 4 d, 8 d and 12 d after treatment (DAT), respectively. Roots samples were harvested at the end of experiment. Treatments were arranged as a completely randomized design and each treatment having four plant-pot systems as four replicates.Four weeks after canopy and root system establishment, plants were separated into three groups  according to the similar transpiration rate calculated based on the weight difference at three-day intervals before salt treatment initiation described by Hu 3)2·4H2O; (3) 300 mM NaCl; (4) H2O2; (5) H2O2 + 300 mM NaCl; (6) 300 mM NaCl- Ca2+ . All reagents were dissolved in half-strength Hoagland’s nutrient solution. Shoot samples were harvested at 0, 12 h, 2 d, 4 d, 8 d and 12 d after treatment, respectively. Roots samples were harvested at the end of experiment.The growing conditions were similar with those of Experiment 1. Plants were separated into six groups with the same method as shown in Experiment 1. The six treatments arranged in a completely randomized block design with four replicates were as follows: (1) nutrient solution (control); (2) 300 mM NaCl + 7 mM Ca was measured using a conductance meter . The samples were then autoclaved at 120 °C for 20 min to completely disrupt the tissues and to release all electrolytes. After cooling to room temperature, the conductance of the incubation solution with killed tissues (Cmax) was determined. Relative EL was calculated using the formula: EL (%) = (Ci/Cmax) × 100.Cell membrane stability was determined by the electrolyte leakage (EL) level measured based on the method described by Hu Turf quality was rated visually based on turfgrass color (percentage green leaves), plant density and degree of leaf wilting on a scale of 0 to 9, where 0 score indicated withered, yellow, thin or dead grass, while 6 indicated minimum acceptable level. A 9 score indicated green, dense and uniform grass2O2 level were conducted according to methods reported previously2O2 and 0.1 mL enzyme extract. CAT activity was determined according to Chance and Maehly2O2 and 0.1 mL enzyme extract was measured at 240 nm absorbance for 1 min. CAT activity was determined through the decomposition of H2O2. APX activity was detected using the method described by Mittler and ZilinskasExtract preparation and assays for SOD (EC 1.15.1.1), CAT (EC 1.11.1.6), POD (EC. 1.11.1.7), APX (EC 1.11.1.11), soluble protein, MDA and H−1cm−1.Cell lipid peroxidation was determined by the MDA content measured as follows. One milliliter of enzyme extract was mixed with 2 ml of reaction solution containing 20% (v/v) trichloroacetic acid and 0.5% (v/v) TBA. The mixture was heated in a water bath at 95 °C for 30 min, then cooled quickly in ice-water bath to room temperature, and centrifuged at 14,000 rpm for 20 min. Absorbance of the supernatant was measured at 532 and 600 nm. MDA content was calculated based on subtracting the absorption at 600 nm from the absorption at 532 nm and calibrated with the extinction coefficient of 155 mM2O2 level was determined based on the method described by Lin and Kao2SO4 (v/v), and then centrifuged at 6000 × g for 15 min at room temperature. The supernatant was measured at 410 nm absorbance. The H2O2 level was calculated according the standard curve generated with known concentrations of H2O2 and calibrated by extinction coefficient of 0.28 μmol−1cm−1.H−1). Subsequently, cDNA synthesis was performed from 2 μg purified RNA using cDNA synthesis kit according to the manufacturer’s protocol . The resultant cDNA was diluted six-fold and kept at −20 °C for RT-PCR analysis.Total RNA extraction was performed from the leaves and roots using Trizol reagent  according to the manufacturer’s instructions and then was incubated with RNase-free DNase . RNA integrity was examined at 260 and 280 nm by spectrophotometer  and checked on a gel electrophoresis in 1.5% agarose gels with 1 μL RNA  and SYBR Green Real-Time PCR Master Mix  in 20 mL reactions. Each reaction mix contained 2 ng of total RNA, 0.5 μL of each primer  and 10 μ2+ was performed by incubating cross-sections of the main root tissue with Fluo 3-AM  according to earlier reports2HPO4, 137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 5 mM glucose, 0.1% BSA, pH 7.4, and incubated at 37 °C for 60 min. And then the preincubated roots were washed with HBSS and fluorescence examination was performed using an IX70 inverted fluorescence microscope , with the following settings: Ex 488 nm, Em 526 nm.Labeling for cytosolic Ca2O2 and O2−, perennial ryegrass roots were stained with 3,3′-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively, based on the method described by Zhang et al.2O2 in perennial ryegrass roots was performed by directly immersion and infiltration of root quarters under vacuum with a freshly prepared DAB solution  at 25 °C in the dark for 30 min. For NBT staining to detect O2− in roots, perennial ryegrass roots were immersed and infiltrated with 3.5 mg mL−1 NBT staining solution in 25 mM KOH-HEPES buffer (pH 7.8) and incubated in the dark for 30 min. After staining, roots were bleached in acetic acid-glycerol-ethanol  solution at 100 °C for 5 min, and then stored in glycerol-ethanol  until photographs were taken. H2O2 as a brown color and O2− as a blue color were visualized.For histochemical detection of H2+, H2O2, NaCl treated leaves and roots were extracted based on the procedure reported by Roessner et al.−1 in chloroform) with 20 μL internal standard (2 mg mL−1 ribitol in water) was added. Extraction was carried out at 70 °C in a metal bath for 15 min (each 5 min vortex 5 s). The tube was centrifuged for 5 min at 12 000 g. The supernatant was decanted to new 10-mL tubes, and 1.5 mL of water and 0.75 mL of chloroform were added. The mixture was vortexed thoroughly and subsequently centrifuged for 15 min at 8000 g. 300 μL polar phase (methanol/water) was decanted into 1.5 mL HPLC vials and dried in a benchtop centrifugal concentrator  overnight. The dried residue was redissolved and derivatized with 80 μL of 20 mg mL−1 methoxyamine hydrochloride in pyridine for 2 h under intensive oscillation (200 rmp) at 37 °C, and followed by a 2 h treatment at 37 °C with 50 μL N-methyl-N- trimethylsilyltrifluoroacetamide (MSTFA).Metabolites from controls, Caet al.−1 in 2 min after injection, and finally held at 260 °C for 10 min. Helium gas was used as carrier set with a constant flow set at 1 mL min −1. The MS measurement were set at electron impact (EI) source, electron energy 70 eV, solvent delay 4 min and the scan range 30–650 m/z at 0.6 scan s−1. Retention time (RT), retention indices  and NIST Mass Spectral Database (version 11) were implemented to identify the target metabolites. Only metabolite detected at least three in five samples was considered true. Each metabolite was finally identified based on the internal consistency of RT and RI. For each sample in controls and Ca2+, H2O2, NaCl treated leaves and roots, we performed four biological replicate and two technical replicates per biological experiment.For GC-MS analysis, a 1 μL of the derivatization solutions was determined with a GC–MS  system based on the method described by Hancock P < 0.05 level.Statistical significance was performed by ANOVA using the Statistics Analysis System (SAS) . Means for physiological measurements, gene expression and metabolites between treatments were separated using the Fisher’s least significant difference test (LSD) at How to cite this article: Hu, T. et al. H2O2 and Ca2+-based signaling and associated ion accumulation, antioxidant systems and secondary metabolism orchestrate the response to NaCl stress in perennial ryegrass. Sci. Rep.6, 36396; doi: 10.1038/srep36396 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Acute myeloid leukemia (AML) is deadly hematologic malignancy. Despite a well-characterized genetic and molecular landscape, targeted therapies for AML have failed to significantly improve clinical outcomes. Over the past decade, proteasome inhibition has been demonstrated to be an effective therapeutic strategy in several hematologic malignancies. Proteasome inhibitors, such as bortezomib and carfilzomib, have become mainstays of treatment for multiple myeloma and mantle cell lymphoma. In light of this success, there has been a surge of literature exploring both the role of the proteasome and the effects of proteasome inhibition in AML. Pre-clinical studies have demonstrated that proteasome inhibition disrupts proliferative cell signaling pathways, exhibits cytotoxic synergism with other chemotherapeutics and induces autophagy of cancer-related proteins. Meanwhile, clinical trials incorporating bortezomib into combination chemotherapy regimens have reported a range of responses in AML patients, with complete remission rates >80% in some cases. Taken together, this preclinical and clinical evidence suggests that inhibition of the proteasome may be efficacious in this disease. In an effort to focus further investigation into this area, these recent studies and their findings are reviewed here. Finally, prognosis is dismal for those who cannot tolerate standard induction chemotherapy, with a median survival of only 5–10 months and OS of <5%.2 Thus, there is a clear and emergent need for the development of new therapeutic approaches for AML.Acute myeloid leukemia (AML) is a lethal hematologic malignancy characterized by the neoplastic accumulation of immature myeloid cells.4 As such, the proteasome has an integral role in a variety of cellular processes, including cell survival, cell signaling and cell-cycle progression.5 Malignant cells are highly dependent on increased protein production and degradation, suggesting that they would be sensitive to proteasome inhibition.8 Indeed, proteasome inhibition is a mainstay of therapy in lymphoid malignancies. Proteasome inhibitors, such as bortezomib and carfilzomib, are now incorporated into standard of care regimens for most patients with multiple myeloma (MM) and other plasma cell neoplasms, and this approach has yielded significantly improved clinical responses and OS for these patients.9 Proteasome inhibition has also shown efficacy in the initial treatment of mantle cell lymphoma (MCL)10 and in the relapsed/refractory setting for other non-Hodgkin lymphomas, such as follicular lymphoma.12One promising molecular target is the proteasome, a large multimeric protein complex that degrades unneeded or damaged proteins.Several pre-clinical and early stage clinical trials investigating the role of the proteasome and proteasome inhibition in AML have shown promising results. In this review, we discuss these studies and their findings.13 In AML, NF-κB is constitutively active in leukemic stem cells (LSCs), but not in normal hematopoietic progenitor cells.13 This constitutive NF-κB activity is supported by autocrine signaling via tumor necrosis factor α (TNF-α), which directs the proteasome-mediated degradation of the NF-κB inhibitor IκBα, thereby liberating cytosolic NF-κB.14 As NF-kB promotes TNF-α expression, a positive-feedback loop is created between NF-κB and TNF-α, promoting cell survival and progression of the leukemia (14Nuclear factor κB (NF-κB) is a transcription factor that promotes cell survival and proliferation and has been implicated in the pathogenesis of numerous malignancies.leukemia .1414 This liberates NF-κB and allows the complex to translocate to nucleus where it can direct the expression of target genes, including TNFα. The importance of the proteasome in this process is underscored by early studies of the effects of proteasome inhibition on NF-κB signaling. For example, treating AML cells with the proteasome inhibitor MG-132 increased the amount of phosphorylated IκBα, leading to a strong inhibition of NF-κB activity, decreased expression of NF-κB gene targets and induction of apoptosis in AML cells (including LSCs).13 Importantly, normal hematopoietic stem cells (HSCs), which are not reliant upon NF-κB activity, were spared from this effect.13 These results were amplified when MG-132 was combined with the anthracycline idarubicin. Treating primary AML cells with this combination ablated LSCs and prevented tumor engraftment in NOD/SCID xenogeneic mouse models, while the ability of normal HSCs to proliferate was not significantly impaired.15This NF-κB/TNF-α feedback loop – and the survival of LSCs – is highly dependent upon the proteasomal degradation of the regulatory protein IκBα. Under normal, unstimulated circumstances, IκBα binds to NF-κB and sequesters it in the cytosol, preventing NF-κB from binding its gene targets within the nucleus. TNF-α signaling drives the phosphorylation of serine residues 32 and 36 on IκBα, leading to ubiquitination and proteasomal degradation.17 When primary AML cells from 28 patients were treated with either bortezomib or conventional chemotherapeutics , bortezomib was found to be the most potent of these agents  while exhibiting less toxicity than doxorubicin towards normal residual lymphocytes.17Because of the poor stability, low bioavailability and relatively low specificity of MG-132, more recent studies have used bortezomib to modulate NF-κB signaling in AML. Like MG-132, bortezomib treatment of AML cell lines and primary blasts increases the abundance of phosphorylated IκBα, reduces NF-κB activity, and triggers apoptosis via permeabilization of the mitochondrial membrane, release of cytochrome C, and cleavage of caspases 3, 8 and 9.in vitro when co-administered alongside a variety of other chemotherapeutics. As anthracyclines  are already mainstays of AML therapy, there have been several studies investigating the effects of co-treatment with these agents.20 By and large, the synergistic effects observed in this setting appear to be due to enhanced inhibition of NF-κB signaling and, to a lesser degree, activation of p53.15 Co-administration with histone deacetylase inhibitors  also perturbs NF-κB, while inhibition of AKT signaling and down-regulation of anti-apoptotic proteins such as Bim, Bcl-2 and XIAP (X-linked inhibitor of apoptosis protein) are also observed.25Bortezomib has been shown to exhibit synergistic anti-AML cytotoxicity (against both cell lines and primary samples) 30 has also been studied. This approach is further supported by evidence that bortezomib treatment indirectly upregulates transcription of DNA methyl-transferase 1 (DNMT1), and azacitidine has been shown to inhibit the activity of DNMT enzymes.29 In addition, bortezomib has been shown to upregulate the expression of the inhibitory microRNA precursor miR-29b, and because patients with elevated miR-29b expression have shown promising responses to decitabine, this combination regimen was also tested clinically.30Bortezomib co-treatment alongside nucleoside analogs 26Taken together, this data suggests that proteasome inhibition remains an efficacious therapeutic mechanism independent of other approaches. Finally, bortezomib retains its activity regardless of p53 status and in treatment-resistant settings (in both cell lines and primary samples), hinting that bortezomib could have a significant role in the relapsed/refractory setting as well.31 It involves the rearrangement of subcellular membranes to sequester organelles or proteins within autophagosomes and subsequent delivery of these species to the lysosome for degradation.31 Under conditions of extreme cellular stress, however, cells can instead use autophagy to degrade essential components and undergo cell death.32 Intriguingly, bortezomib-induced proteasome inhibition has been shown to induce autophagy in both primary AML samples and several cell lines  are found in ∼20% of AML patients and are associated with poorer outcomes.et al.31 showed that FLT3-ITD+ primary AML samples are more sensitive than wild-type samples to proteasome inhibition by bortezomib, and that this sensitivity correlates to FLT3-ITD allele burden. In this setting, bortezomib treatment led to inhibition of mammalian target of rapamycin complex 1 , increased conversion of LC3-I–LC3-II (a marker of autophagosome formation), and ultimately, autophagy of FLT3 protein.31 Furthermore, autophagy of FLT3 occurred in both quizartinib-sensitive and resistant cells lines, indicating that bortezomib treatment may represent a therapeutic strategy in FLT3-ITD+ AML patients refractory to tyrosine kinase inhibition.31Larrue miR-146a, a candidate gene in del(5q) MDS/AML that is reduced significantly in both del(5q) and normal karyotype cases.32 Treating AML cell lines with bortezomib induced autophagy of TRAF6 and subsequent apoptosis.32 Although TRAF6 overexpression conferred some resistance to bortezomib, sensitivity could be rescued by RNAi-mediated knockdown of TRAF6.32 Subsequent gene-expression analyses found that TRAF6 regulates the expression of the PSMA1 gene, which encodes the α1 subunit of the proteasome.32 This demonstrates that while TRAF6 is regulated by bortezomib-induced autophagy, it also alters sensitivity to the drug by controlling PSMA1 expression.32TNF receptor associated factor 6 (TRAF6) is a key protein target of 33 Second, overexpression of proteasome subunits, such as PSMA1, confers resistance to bortezomib.32 Here, knockdown of upstream elements controlling the expression of PSMA1, including TRAF6, restored sensitivity in drug-resistant cell lines.32 Third, some in vivo data suggests a physical barrier to bortezomib may exist. In an MllPDT/wt:Flt3ITD/wt murine model – which develops spontaneous AML with phenotypic similarities to the human disease – bortezomib treatment alone did not produce a response in vivo, despite evidence of ex vivo efficacy.34 When encapsulated within liposomes, which is predicted to enhance tumor permeability and retention, bortezomib was able to induce a long-term disease-free remission in 80% of the mice.34Unsurprisingly, several mechanisms of resistance to the cytotoxic effects of proteasome inhibition have been noted. First, cells (including LSCs) can overcome the effects of bortezomib by upregulating levels of NF-κB and accumulating the anti-apoptotic proteins MCL-1 and Bim. In these cases, co-administration of mechanistically relevant agents, such as the MCL-1 inhibitor obatoclax, re-sensitized the cells to bortezomib treatment.+ cells, despite similar overall cytotoxic efficacy.35 In these cases, the second-generation proteasome inhibitor carfilzomib demonstrated superior reduction of LTC-IC frequency, perhaps due to the irreversible, more specific, and more prolonged inhibition of the proteasome. Congruent with other studies demonstrating the selectivity of proteasome inhibition, neither the LTC-IC frequency of normal CD34+ cells nor the colony-forming potential of normal bone marrow cells were significantly affected by carfilzomib exposure, implicating a leukemia-specific effect.35 Finally, this study also showed that, similar to bortezomib, treatment with carfilzomib upregulates MCL-1 expression, conferring treatment resistance that could potentially be overcome by simultaneous administration of obatoclax.35Another mechanism of resistance may be inherent to the drugs themselves. Some studies have suggested that bortezomib has limited efficacy in reducing the long-term culture-initiating cell (LTC-IC) frequency of some primary AML CD34Pgp, MRP-1, BRCP and LRP seems to have little, if any, effect on the efficacy of proteasome inhibition with bortezomib in AML cell lines overexpressing these proteins.26Encouragingly, expression of multi-drug resistance genes such as NPM1) gene mutations represent the most frequent genetic lesion in AML, present in ∼30% of cases.1 In primary NPM1-mutated AML cells, co-treatment with arsenic trioxide and all-trans retinoic acid – a regimen typically reserved for acute promyelocytic leukemia – was shown to induce proteasome-dependent degradation of NPM1 leukemic protein and induce apoptosis.36 Furthermore, this degradation of NPM1 was shown to potentiate the response to daunorubicin, which was unexpected considering that proteasome inhibition has been shown to be synergistic with anthracycline therapy in other cases of AML.36Despite the promising prospect of proteasome inhibition in the treatment of AML, other therapeutic regimens rely upon a functioning proteasome for their efficacy. Nucleophosmin 1  of the proteasome have been examined as potential clinical markers for a variety of outcomes, including sensitivity to treatment regimens and for OS. Matondo 38 To provide a more rapid clinical assessment of proteasome status, Ma et al.38 sought to correlate levels of circulating proteasome complexes to a variety of clinical and laboratory data. They showed that the levels and cumulative activity of circulating proteasomes were significantly higher in AML patients than healthy controls; however, when the proteasome activity was normalized to the levels of proteasome protein in the samples, the chymotrypsin-like (ChTL) unit activity was lower in AML samples. This result correlates to in vitro work showing that KG1a AML cells, which are highly sensitive to bortezomib treatment, overexpress the 20S cCP but have lower ChTL activity due to altered expression of the 19S regulatory complex.37 Therefore, the increased activity of circulating proteasomes is likely a measure of leukemic tumor burden rather than enhanced catalytic efficiency. Indeed, levels of circulating proteasomes were predictive of ubiquitin levels, lactate dehydrogenase concentration and white blood cell count.38Circulating proteasomes behave similarly to and most likely reflect the activity of cellular proteasomes in primary leukemia samples.38 Similarly, lower proteasome levels were a strong predictor of survival in AML patients with unfavorable and intermediate cytogenetics, and this remained true independent of other major risk factors examined .38 However, the duration of CRs did not correlate with either proteasome or ubiquitin levels.38 Ultimately, these studies suggest that circulating proteasome levels and enzymatic activities may prove to be useful biomarkers in risk-stratifying AML patients and in identifying patients who may be more likely to respond to proteasome inhibition.AML patients with circulating proteasome levels below the median level (875 ng/ml) were found to have significantly better response rates to current treatment regimens than those with higher levels.39 Although its primary function is to degrade antigenic peptides for major histocompatibility complex class I (MHC-I) restricted antigen presentation, the i-prot has also been shown to influence T-cell polarization and differentiation, cytokine production by macrophages, degradation of proteins damaged by oxidative stress and NF-κB signaling.41 As such, the i-prot is suspected to have a substantial role in the pathogenesis of a broad range of disorders, including inflammatory autoimmune diseases and hematologic malignancies.The immunoproteasome (i-prot) is an inducible proteasome primarily recognized for its role in antigen processing and presentation.41 The i-prot is assembled when components of the c-prot are replaced by i-prot-specific subunits (42 suggesting that the i-prot degrades a selective subset of polyubiquitinated proteins.In contrast to the constitutive proteasome (c-prot), which is tonically expressed in most cells, the i-prot is formed in response to the inflammatory cytokines TNF-α and interferon-γ (IFN-γ).subunits ; these sLMP2 (PSMB9), LMP10 (PSMB10) and LMP7 (PSMB8), respectively, and their expression is induced at least partly by TNF-α and IFN-γ.3 When incorporated into the iCP, these distinct subunits confer a different profile of substrate specificity that helps shape the diverging roles of the i-prot and c-prot; for example, the i-prot preferentially hydrolyzes proteins following non-polar amino acids, thereby generating peptide sequences with hydrophobic C-termini that are readily suited for incorporation into MHC-I complexes.3 Furthermore, the unique composition of the iCP has enabled the pre-clinical development of small molecule inhibitors specific for the iCP, which have served as valuable tools for probing the role of the i-prot in a variety of disease models.Specifically, the core particle of the immunoproteasome (iCP) is formed when the three main catalytic subunits of the cCP – β1c, β2c and β5c – are replaced by subunits specific to the i-prot, β1i, β2i and β5i. These proteins are encoded by the genes LMP2 in particular – have been associated with an increased risk of developing AML, perhaps due to alterations in the substrate specificity or proteolytic activity of the complex.43 Second, the upregulation of total proteasome machinery that is observed in AML cells is characterized by a marked overexpression of the iCP, outweighing the cCP by approximately threefold.44 The ratio of β5i/β5c is even higher (7.9-fold).44 Third, there is recent evidence that the i-prot has a crucial role in mediating mTORC1-driven cell survival.45 Notably, mTORC1 hyperactivation is observed in many cell growth disorders, including AML. It was shown that PRAS40 (proline-rich Akt substrate 40 kDa), a component of mTORC1, binds to the β1i and β5i subunits and sequesters them to hinder iCP formation.45 Once phosphorylated by mTORC1, PRAS40 releases the iCP subunits, permitting their association with the chaperone protein POMP (proteasome maturation protein) and thereby facilitating i-prot assembly.45 In this manner, activation of mTORC1 serves to upregulate i-prot activity, enabling cells to deal with the defective ribosomal products that accumulate alongside increased protein synthesis and cell division. Furthermore, this mechanism has been validated in AML, thus defining a novel mechanism by which these cells protect themselves against protein stress and further elucidating a potentially fruitful therapeutic target.45The i-prot is an especially interesting drug target for the treatment of AML for several reasons. First, somatic mutations in genes encoding the iCP subunits – in vitro preparations of human proteasome proteins, PR-957 was shown to selectively target β5i with up to 15-fold greater selectivity for the iCP than the cCP.46 Further, PR-957 was shown to inhibit β5i-specific antigen presentation and reduce the production of several cytokines, including interleukin (IL) 23, IL-6, IL-2, TNF-α and IFN-γ, thereby modulating the early activation of T cells and their differentiation into inflammatory effector cells.46In vivo, PR-957 and a related ketoepoxide, PR-924, have shown efficacy in murine models of rheumatoid arthritis and MM, respectively, with both agents being well tolerated.47Perhaps the most widely used i-prot inhibitor is PR-957  occurring at doses of 1.5 mg/m2. Proteasome inhibition was observed to be dose-dependent, reaching a maximum value of 68% at 1.5 mg/m2. This peak inhibition was observed 1 h after treatment, and proteasome activity returned to baseline over 72 h. In addition, peripheral blood mononuclear cells isolated from these patients underwent significant (>50%) apoptosis when exposed to bortezomib in vitro. Clinically, five patients met criteria for hematological improvement, four for a decrease in blast count (to ⩽5% in three patients) and one for an improvement in neutrophils. The primary diagnoses  for these patients were not reported.With FDA-approved indications for MM and MCL plus pre-clinical efficacy in numerous other lymphoid malignancies, bortezomib has been incorporated into numerous clinical trial regimens either as a single agent or in combination with other drugs . An earl49 including 14 patients with either untreated or relapsed/refractory AML, single-agent bortezomib was given at 1.5 mg/m2 twice weekly for 2 weeks in a 21-day cycle. A reduction of peripheral and/or bone marrow blasts was observed in 8 of the 13 evaluable patients (61%), and the median OS was 4 months (range 0.25–10 months). Peripheral neuropathy was the most frequently reported adverse event, experienced by 7 of the 13 patients (54%); 4 of these 7 (57%) had to discontinue treatment because of the neurotoxicity.In a phase II study (EUDRACT 2006-006923-38)20 administered bortezomib twice weekly at escalating doses  alongside once weekly doses of idarubicin (10 mg/m2) for 4 weeks. Twenty patients were treated, 13 elderly individuals (median age 68 years) with newly diagnosed AML and another 7 with relapsed AML (median age 58). The study found the maximum tolerated dose (MTD) for this regimen to be 1.2 mg/m2 bortezomib plus 8 mg/m2 idarubicin, with common adverse events including neutropenic fever, infections, constitutional symptoms and gastrointestinal symptoms. Unlike the single-agent studies, no patients experienced neurotoxicities. One patient died due to treatment-related infection, and another four died from refractory AML while on the study. Ultimately, only four (20%) patients achieved a CR, though most (75%) did experience a hematologic response in the form of reduced peripheral blasts. The median OS in this high-risk group of patients was 4 months.A phase I study (NCT00382954)18 of bortezomib plus pegylated liposomal doxorubicin (PegLD) enrolled 42 patients, five of whom were diagnosed with AML. Bortezomib was administered in a similar fashion as the single-agent trial and the same MTD of bortezomib was identified , likely due to the distinct mechanisms of action and lack of interactions between the two agents. Still, the dose-limiting toxicities observed were reminiscent of both agents, including cytopenias, fatigue, neuropathy and diarrhea. Of the five AML patients who enrolled, only two received two or more cycles of the treatment. The first of these two patients had newly diagnosed AML, but was not a candidate for standard therapies; they achieved a partial remission (PR) after two cycles, with a reduction in peripheral blood absolute blast count from 2,688 μl−1 (7%) at baseline to 107 μl−1 (1%). The second AML patient had relapsed disease post several induction regimens and an autologous hematopoietic stem cell transplantation; after two cycles, the hypercellular bone marrow with 19% blasts became normocellular with 4% blasts, though a clonal cytogenetic abnormality was still identified. This patient's treatment was then interrupted due to disseminated Varicella zoster reactivation, and upon resolution, the leukemia had progressed. Additional cycles of the bortezomib/PegLD regimen again converted their hypercellular marrow with 28% involvement to a normocellular marrow with only 5% blasts.Another phase I trial29 a phase I study (NCT00624936)50 investigated the combination of daily azacitidine (75 mg/m2) and escalating bortezomib (up to 1.3 mg/m2) in 23 adults with relapsed/refractory AML. Although these doses were reached without limiting toxicities, several patients did experience febrile neutropenia and/or infection. Five of the 23 patients (22%) achieved a remission, with two CRs and three CRs with incomplete recovery of platelets or neutrophils (CRi). Furthermore, four of these five responders presented with cytogenetic abnormalities (various karyotypes), and three of them achieved a cytogenetic CR.Building upon pre-clinical data,51 In this phase I trial (NCT00703300),51 19 poor-risk AML patients (median age 70 years) were treated with an induction regimen of 10 mg/m2 decitabine intravenously on days 1–10. This was augmented with bortezomib escalated up to 1.3 mg/m2 on days 5, 8, 12 and 15. Although this dosing schedule was tolerable, bortezomib-related neuropathy was noted after repetitive cycles (none during cycle 1). Among the previously untreated patients, 5 of 10 (50%) achieved a CR (n=4) or CRi (n=1), while the overall CR/CRi rate was 7 of 19 (37%).Similarly, the combination of bortezomib and decitabine was also explored.52 exploring the combination of bortezomib with the histone deacetylaseinhibitor vorinostat in high-risk patients with AML (n=8) or MDS (n=4) was terminated after three fatal cardiac events. The significant toxicity observed in this trial was quite unexpected given the favorable profile of similar studies in patients with MM and MCL. However, this patient population was heavily pretreated with a median of three prior regimens, and over half were relapsed after allogeneic hematopoietic stem cell transplantation. Although performance statuses were reasonably high, the extensive pretreatment may have affected the patients' ability to tolerate further toxicity. In the end, only one patient – an AML patient who had failed prior induction therapy – achieved CR; they remained transfusion independent for ∼6 months before evidence of disease progression. All patients succumbed to progressive disease within 1 year.Despite pre-clinical efficacy, a phase II trial (NCT00818649)19 investigated the combination of bortezomib with a standard of care induction regimen (idarubicin plus cytarabine). A total of 31 patients were enrolled, 9 with relapsed AML and the other 22 with previously untreated AML. Non-hematologic toxicities included hypoxia, hyperbilirubinemia, transaminitis, fatigue and diarrhea. Overall, 19 (61%) patients achieved a CR, another 3 (9%) achieved a CR without recovery of platelets (CRp) and an additional 2 (6%) experienced a partial response (PR). For the 22 patients who achieved CR/CRp, the median disease-free survival was 15.3 months and the median OS was 17.6 months.Another phase I study (NCT00505700)28 by the same investigator, the combinatorial use of bortezomib plus daunorubicin and cytarabine in 95 adults with previously untreated AML was examined. The results were similar, with 62 of 95 patients (65%) achieving a CR and an additional 4% achieving a CRp. Significant (grade 3 or greater) neuropathy was reported in 11 (12%) patients. Median disease-free survival was 8 months, whereas OS was 12 months.In a phase II study (NCT00742625)53 In 23 relapsed/refractory AML patients who received this five-drug regimen, the overall CR/CRi rate was 82.5%, with 13 patients (56.5%) achieving CR and another 6 (26%) achieving CRi; the median OS for all patients was 330 days. Treatment-related toxicities were as expected, including peripheral neuropathy, decreased ejection fraction and diarrhea.Bortezomib was also examined alongside the tyrosine kinase inhibitor midostaurin plus mitoxantrone, etoposide and cytarabine (MEC) in a phase I trial (NCT01174888).54 of bortezomib combined with one of two reinduction chemotherapy regimens (either idarubicin/cytarabine or cytarabine/etoposide) in children with relapsed, refractory, or secondary AML was terminated due to failure to reach predetermined efficacy thresholds. However, this may be due to how the study defined its efficacy goals. For instance, only CR and CRp were considered efficacious responses, whereas CRi  was excluded. Because the number of patients achieving a CRi (n=5) was similar to those achieving a CRp (n=3), this may have skewed the results at the interim analysis and led to the early trial cessation. In this study, overall CR rates reached 57% with a two-year OS of 39±15%. Correlative studies in this population revealed that depletion of leukemia-initiating cells in the bone marrow after one cycle of treatment was significantly associated with a clinical CR.Again, not all trials have been successful. A phase II study (NCT00666588)58 A phase I dose escalation study (NCT00580242)59 evaluated the MTD of bortezomib  when added to standard dose lenalidomide (10 mg/day PO for the first 21 days) in a 28 day cycle. Twenty three patients were enrolled, nine of whom had AML (other 14 had MDS). The MTD of bortezomib in this regimen was 1.3 mg/m2, and among the nine AML patients, there was one CR and a median OS of 5.3 months.Lenalidomide is an immunomodulatory agent with anti-angiogenic properties that has demonstrated efficacy in MM, MDS and MCL.FLT3 or BCR-ABL mutations and inducing apoptosis in primary AML cells.62 In a phase I study (NCT00103272)63 of 17-AAG plus bortezomib in 11 patients with relapsed/refractory AML, the MTDs were 150 mg/m2 and 0.7 mg/m2, respectively. Hepatic and cardiac toxicities were dose limiting, and no clinical benefit was observed.The heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) has demonstrated pre-clinical efficacy in AML, exhibiting cytotoxic effects in AML cell lines harboring Overall, the clinical use of proteasome inhibitors in AML appears to offer favorable outcomes, especially when combined with other combination chemotherapy regimens. As such, there are several currently active and recruiting trials continuing to explore the role of proteasome inhibition in AML . AlthougThese studies highlight the important role of the proteasome in AML biology and suggest that proteasome inhibition may be an effective therapeutic option in AML. To better understand the molecular effects and clinical outcomes of proteasome inhibition, further investigation is needed is several areas.64 As proteasome inhibitors have demonstrated cytotoxic efficacy against LSCs, further study regarding how this approach can be targeted towards these cells may lead to more efficacious clinical use of these agents.15 One potential avenue for accomplishing this is through selective inhibition of the i-prot. Given that somatic mutations within the i-prot have been associated with an increased risk for developing AML and that iCP subunits are robustly overexpressed in primary AML cells, the i-prot is an attractive target for further drug development.45 Whether or not i-prot inhibition is more efficacious than c-prot inhibition (and whether any efficacy holds for LSCs), however, remains to be seen. Third, further work is needed to identify more rational drug combinations based upon known AML biology. For example, the addition of the MCL-1 inhibitor obatoclax to bortezomib was able to overcome resistance to proteasome inhibition and restore sensitivity to this agent.33 Likewise, there is evidence suggesting that a similar approach would be efficacious with the second-generation proteasome inhibitor carfilzomib.35 Finally, the identification of leukemia subtypes in which proteasome inhibition is likely to have a beneficial effect is of high priority. Like most therapies, it is unlikely that proteasome inhibition will be universally efficacious, so the ability to rapidly identify patients who will benefit from this intervention would be immensely helpful and enable the responsible use of these agents.First, the eradication of the primitive LSCs has been a goal of the field since their elaboration over two decades ago.Ultimately, the proteasome has proven to be a highly interesting and potentially useful therapeutic target for AML. Current work has begun to elaborate its specific role in the biology of the disease, and the clinical outcomes of proteasome inhibition are encouraging.


Ca2+ is a second messenger regulating a variety of cellular processes. We hypothesized that the compound, celastrol, affecting cytosolic Ca2+ mobilization could serve as a novel strategy to combat RA. To address this issue, celastrol was used as a molecular tool to assay the inflammatory gene expression profile regulated by Ca2+. We confirmed that celastrol treatment mobilized cytosolic Ca2+ in patient-derived RASFs. It was found that 23 genes out of 370 were manipulated by Ca2+ mobilization using an inflammatory and autoimmunity PCR array following independent quantitative PCR validation. Most of the identified genes were downregulated and categorized into five groups corresponding to their cellular responses participating in RA pathogenesis. Accordingly, a signaling network map demonstrating the possible molecular circuitry connecting the functions of the products of these genes was generated based on literature review. In addition, a bioinformatics analysis revealed that celastrol-induced Ca2+ mobilization gene expression profile showed a novel mode of action compared with three FDA-approved rheumatic drugs . To the best of our knowledge, this is a pioneer work charting the Ca2+ signaling network on the regulation of RA-associated inflammatory gene expression.Rheumatoid arthritis synovial fibroblasts (RASFs) are fundamental effector cells in RA driving the joint inflammation and deformities. Celastrol is a natural compound that exhibits a potent anti-arthritic effect promoting endoplasmic reticulum (ER) stress mediated by intracellular calcium (Ca Rheumatoid arthritis (RA) is the most common chronic systemic autoimmune inflammatory disease. RA affects around 1% of people worldwide, and is more prevalent in women , glucocorticoids (steroid hormones), and disease-modifying antirheumatic drugs (DMARDs) are standard pharmaceutical interventions for the inflammatory disorder. Of note, DMARDs medications suppress immune system functions to prevent further structural damage of bone and cartilage in the affected joints  is a traditional remedy for RA treatment  mobilization  and cancer . Medium was changed every 3 days and, after 2 weeks, the synovial tissues were removed from the cultured medium. RASFs were digested by 0.25% trypsin for 5 min at 37°C. The cell suspension was diluted with DMEM containing 20% FBS and 1% Penicillin/Streptomycin with L-Glutamine (PSG), and separated into other flasks. Cell cultures were maintained at 37°C in a humidified incubator (atmosphere of 5% CO2). The purification of RASFs was validated by staining CD90. Cultured RASFs from passages 5–7 were employed for the below analysis. Unless otherwise specified, all reagents were purchased from Sigma-Aldrich .Primary culture RASF were isolated from synovium obtained from RA patients who had undergone knee surgery for synovectomy. Diagnosis of RA in these patients was made according to the American Rheumatism Association's 1987 revised criteria for classification of RA , which contains a proprietary Ca2+-sensitive fluorophore, according to the manufacturer's instructions. In brief, 10000 RASFs per well were seeded in black wall/clear bottom 96-multiwell plates from Costar  and cultured for 24 h before treatment. After that, calcium 6 reagent was added directly to cells, and cells were incubated for an additional 2 h at 37°C and 5% CO2. One micromolar of celastrol  was then added to the wells and immediately subjected to data acquisition on the FLIPR Tetra High-Throughput Cellular Screening System  at room temperature using a 1-s reading interval throughout the experiments.Intracellular cytosolic Ca5 RASFs cells were cultured in 35 mm confocal disc at 37°C CO2 incubator for 24 h. FLIPR Calcium 6 reagent was added to cells at 37°C for 30 min. RASFs were then washed 3 times with HEPES buffer saline and incubated at 37°C in an imaging chamber for another 10 min. Changes in cytosolic [Ca2+] levels were monitored by following changes in FLIPR Calcium 6 fluorescence upon addition of 1 μM celastrol in HBSS buffer, using the real-time mode for 4 min by epifluorescence microscopy . Data Inspection Program provided by the DeltaVision software was used to measure the intensity of the FLIPR Calcium 6 fluorescence and the mean fluorescence intensity was monitored at 525 nm and plotted against time (s).2 × 10RNA was extracted using RNeasy Mini Kit  from RASFs untreated (control), treated with celastrol [1 μM], or celastrol in the presence of BAPTA/AM [10 μM]  for 24 h. RNA concentration was determined using the NanoDrop 2000c Spectrophotometer (Thermo Scientific) and 1 μg of RNA was used to synthesize cDNA with RT2 First Strand Kit . Three independent biological samples were employed.2 Profiler™ PCR Array Human Inflammatory Response & Autoimmunity 384HT kit  was assessed according to the manufacturer's instructions. This PCR array contains primers for 370 genes related to various functions of the immune system, from cytokines/chemokines to their receptors, cytokine production, and other proteins and receptors involved in acute-phase, inflammatory and humoral immune responses. Analysis of results was achieved using the integrated web-based RT2 Profiler™ PCR Array Data Analysis software from Qiagen, which calculated all ΔΔCt-based fold-change and fold-regulation from three independent raw data. Validation of the identified gene expression pattern upon celastrol-induced calcium mobilization was performed by quantitative PCR with self-designed primers . Primer sequences  and ViiA™ 7 Real Time PCR System (Applied Biosystems). The RT2+ mobilized genes in this report were searched for their implication in Ca2+-dependent function using NCBI database and KEGG pathways. Furthermore, NCBI database (Geo DataSets) was searched for datasets that were generated by treating RA patient with FDA-approved drugs. Three studies were selected that evaluated Rituximab (GDS4903), Tocilizumab (GDS5068), and Methotrexate (GDS5069) for RA treatment. The gene expression profile was transformed into fold change (2−ΔΔCT method) and merged with our dataset (celastrol-mediated Ca2+ mobilization regulated genes) using R (3.3.2). Merged datasets was then processed with Phyloseq (1.19.1) and ggplot2 (2.2.1) packages to generate principal coordinate plot with weighted UniFrac distance. Additionally, SIMPER analysis was performed with R (3.3.2) using vegan (2.4-3) to determine genes that are similarly responding to celastrol [Ca2+] and FDA-approved drugs .The identified celastrol-mediated Ca2+ mobilization in RASFs, intracellular cytosolic Ca2+ dynamic and single live-cell Ca2+ imaging was performed on celastrol-stimulated RASFs. As shown in Figures 2+ dynamic changes in RASFs within c.a. 30 s, confirming that celastrol is a suitable tool for Ca2+-flux effects studies. Quantitative PCR was performed to identify the inflammatory genes expression profile affected by celastrol-mediated Ca2+ mobilization with the use of RT2 Profiler™ PCR Array Human Inflammatory Response & Autoimmunity. Only those genes with changes in fold regulation above ±1.5-fold in the PCR array result were considered to analyse in this study. After celastrol treatment, the expression of 134 out of the total 370 examined genes were up-regulated or down-regulated Figure 2+ in the expression of these genes. In the presence of Ca2+ chelator, BAPTA/AM, we identified that 72 out of the 134 genes were regulated by celastrol-mediated Ca2+ mobilization. In addition, those genes which commenced their expression at Ct ≥ 30 were excluded decreasing the number of validated genes to 40 Figure 2+-dependent manner except CD40 which was upregulated in response to the changes in cellular Ca2+ level , calpastatin (CAST), and Toll-like receptor 6 (TLR6), participate in all of the above described cellular functions. Among these genes, 17 of them play a role in apoptosis/cell death, 17 genes regulate cell migration/invasion, 15 genes are involved in angiogenesis, 22 genes can promote or inhibit cell proliferation, and 21 genes are associated with immunity/inflammation. The products of these genes represent a great variety ranging from upstream cellular receptors, e.g., TLR6 and leptin receptor (LEPR), signaling molecules like cytokines and enzymes, e.g., IK cytokines (IK) and BMP1, to downstream transcriptional factors, e.g., nuclear factor of activated T-cells c3 (NFATC3) and signal transducer and activator of transcription 3 (STAT3). In addition, apart from the genes CAST, CD40, NFATC3, and TRAP1, which had previously been found to be regulated by Ca2+-signaling machinery  calpastatin (CAST)/calpains   of RA patients with methotrexate, rituximab, and tocilizumab. The datasets were subset to the list of genes that matched with celastrol-[Ca2+] regulated genes and comparatively analyzed using UniFrac distance analysis. As shown in Figure 2+] differentially modulated the genes expression. However, celastrol-[Ca2+] regulated genes were comparable with changes observed after methotrexate and tocilizumab treatments, whereas, greater dissimilarity was observed between rituximab and celastrol-[Ca2+]. Moreover, the genes that responded similarly to celastrol-[Ca2+] and methotrexate  model , inositol trisphosphate receptor (InsP3), and ryanodine receptor (RyR) which located on the ER are the key managers of intracellular Ca2+ storage. In addition, the sodium-calcium , together with, other Ca2+ channels such as voltage-operated calcium channel (VOCC), receptor-operated calcium channels (ROCC), and store-operated channels (SOCC), help to control the Ca2+ trafficking between cytoplasmic and extracellular environment. The observed Ca2+ flux in our RASFs is most probably due to the coupling of celastrol with SERCA (unpublished data) which further antagonizes the function of the receptor. The inactivation of SERCA stops the transportation of Ca2+ into the ER and results in cytosolic Ca2+ accumulation which mediates the expression of the validated genes. Upon celastrol treatment, CAST, LEPR, TOLLIP, and TRAP1, could be partially restored to the untreated level by BAPTA/AM implying that other unknown molecular mechanisms may underpin the regulatory machinery. After extensive literature review, the transcriptional regulations of CD40, TRAP1, NFATC3, and CAST, have been reported before which are related to cytosolic Ca2+ mobilization  the calpains/calmodulin-calcineurin-NFATc3 axis; (2) PI3K-Akt/PKC-NF-κB axis; (3) JAK-Stat axis; (4) ERK-MMP axis; and (5) MyD88-AP1 axis. The calpains/calmodulin-calcineurin-NFATc3 axis is per se a well-known Ca2+-associated pathway inevitably regulating the cartilage catabolism and osteoclast formation, angiogenesis, and inflammation during RA pathogenesis  , CD40, NFKB1, LEPR, IL4R, FGF10, and ERBB2, potentially target this signaling axis with most of them functioned molecularly via PI3K, the upstream kinase of Akt and PKC. NF-κB involved extensively in the development of chronic inflammation  and the associated molecular mechanisms underlying the corresponding signaling, therefore, deserve in-depth investigation for completing the signaling network as depicted in this study.The molecular pathways which are mediated by the products of our validated genes may form a complex network in RASFs with the constituting pathways crosstalk with each other. We accordingly generated a blueprint outlining such potential molecular circuitry using literature information documenting the signaling cascades that connect the products of individual validated gene to Ca2+ homeostasis is an important factor regulating the expression of a group of genes in RASFs which may significantly affect RA pathogenesis. As supported by the bioinformatics analysis, this Ca2+-dependent mechanism induced by celastrol is a new mode of action which has not been documented previously by using other conventional RA pharmaceutical interventions such as methotrexate, rituximab, and tocilizumab (Burmester and Pope, BMP1 and ERBB2 which support the therapeutic potency of celastrol in RA treatment. In addition, products of the celastrol induced Ca2+-mediated genes are participating in both upstream and downstream of molecular mechanisms leading to a comprehensive signaling control in a top-down basis not just targeting a single signaling messenger within a particular pathway. Therefore, our data highlighted the potential application of compounds, like celastrol, which are capable of regulating cytosolic Ca2+ level, as molecular tools for investigating the pathomechanisms of RA and other chronic inflammatory disorders. Our findings also provide forecasting platform for the establishment of functional experiments to validate our recent findings by using in vitro and in vivo models which facilitate the exploitation of novel pharmaceutical targets and therapeutic compounds.Taken together, we have discovered that cellular CaIdSRD and SM: conducted the experiments and drafted the manuscript. FG-M: prepared the figures and, materials and methods. IK and WH: conducted the bioinformatics analysis. BL: prepared the discussion and revised the whole manuscript. VW and LL: conceived the idea and designed the experimental plan.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Can a model incorporating patient-specific (comorbidities and age) and acute myeloid leukemia (AML)-specific features  predict mortality after AML treatment?In a multicenter cohort study of 1100 patients, we demonstrated that (1) comorbidities had a significant impact on 1-year mortality after initial therapy for AML, (2) an augmented hematopoietic cell transplant–comorbidity index (HCT-CI) was the best suited index for comorbidity evaluation in AML, and (3) an AML composite model of augmented HCT-CI, age, and cytogenetic/molecular risks has a strong AUC of 0.76 for 1-year mortality.An AML composite model can guide decision-making about treatment of AML. To our knowledge, this multicenter analysis is the first to test and validate (1) the prognostic impact of comorbidities on 1-year mortality after initial therapy of acute myeloid leukemia (AML) and (2) a novel, risk-stratifying composite model incorporating comorbidities, age, and cytogenetic and molecular risks.To accurately estimate risks of mortality by developing and validating a composite model that combines the most significant patient-specific and AML-specific features.P < .10 constructed a multivariate Cox proportional hazards model in which the impact of each covariate was adjusted for that of all others. Then, the adjusted hazard ratios were used as weights. Performances of models were compared using C statistics for continuous outcomes and area under the curve (AUC) for binary outcomes.This is a retrospective cohort study. A series of comorbidities, including those already incorporated into the hematopoietic cell transplantation–comorbidity index (HCT-CI), were evaluated. Patients were randomly divided into a training set (n = 733) and a validation set (n = 367). In the training set, covariates associated with 1-year overall mortality at a significance level of Initial therapy for AML.Death within 1 year after initial therapy for AML.A total of 1100 patients, ages 20 to 89 years, were treated for AML between January 1, 2008, and December 31, 2012, at 5 academic institutions specialized in treating AML; 605 (55%) were male, and 495 (45%) were female. In the validation set, the original HCT-CI had better C statistic and AUC estimates compared with the AML comorbidity index for prediction of 1-year mortality. Augmenting the original HCT-CI with 3 independently significant comorbidities, hypoalbuminemia, thrombocytopenia, and high lactate dehydrogenase level, yielded a better C statistic of 0.66 and AUC of 0.69 for 1-year mortality. A composite model comprising augmented HCT-CI, age, and cytogenetic/molecular risks had even better predictive estimates of 0.72 and 0.76, respectively.In this cohort study, comorbidities influenced 1-year survival of patients with AML, and comorbidities are best captured by an augmented HCT-CI. The augmented HCT-CI, age, and cytogenetic/molecular risks could be combined into an AML composite model that could guide treatment decision-making and trial design in AML. Studying physical, cognitive, and social health might further clarify the prognostic role of aging. Targeting comorbidities with interventions alongside specific AML therapy might improve survival. This cohort study examines the prognostic association of comorbidities on 1-year mortality after initial therapy of acute myeloid leukemia and a risk-stratifying composite model that incorporates comorbidities, age, and cytogenetic/molecular risks. Acute myeloid leukemia (AML) is the most common form of acute leukemia,Mortality in AML, in part, reflects its inherent resistance to therapy and, in part, the deleterious, and at times, lethal effects of treatment. Quantifying both of these risks is critical to the fundamental decision of whether patients should receive any specific therapy and, if so, whether it should be less or more “intense.” Therapy guidelines, such as those of the National Comprehensive Cancer Network (NCCN), have tended to focus largely on an arbitrary age cutoff of 60 or 65 years as the arbiter of the appropriateness of therapeutic intensity.We have shown that accounting for comorbidities can provide valuable information on the risks associated with allogeneic hematopoietic cell transplantation (HCT).We followed the Enhancing the QUAlity and Transparency of Health Research (EQUATOR) reporting guidelines using the Transparent Reporting of a Multivariable Prediction Model for Individual Prognosis Or Diagnosis (TRIPOD) criteria.This is a retrospective hospital-based cohort study of data collected by review of electronic medical records and computer databases for 1100 patients. All patients were consecutively and concurrently treated at each of the AML specialty centers between January 1, 2008, and December 31, 2012. Data were randomly divided into a training set (n = 733) and a validation set (n = 367) for development and validation of the model, respectively. This protocol was approved by the institutional review boards of the Fred Hutchinson Cancer Research Center (FHCRC) and the collaborating sites. All data regarding treatment and demographic information were utilized according to the Declaration of Helsinki. Information was originally recorded and collected for medical purposes; therefore, consent was waived by the institutional review boards of the collaborating institutions.Five academic sites with designated inpatient and outpatient facilities for treatment of AML contributed to this study under the coordination of the FHCRC. The other 4 collaborating sites were Cleveland Clinic, Cleveland, Ohio; Massachusetts General Hospital, Boston; Stanford University, Stanford, California; and the University of Utah, Salt Lake City. Inclusion criteria were (1) age 20 years or older, (2) newly diagnosed non-M3 AML, and (3) initial treatment given during the study period defined herein. Patients receiving palliative care only were excluded from the data set. The different regimens used to treat these patients were classified as low, intermediate, or high intensity as indicated in eTable 1 in the Death within 1 year from initial therapy was the outcome of interest because it includes both early deaths due to regimen-related toxic effects or to lack of response and/or relapse of AML,P < .10 were used to construct a multivariate Cox proportional hazards model in which the impact of each comorbidity or covariate was adjusted for that of all others. To develop both a new AML-specific comorbidity index and a composite multirisk factor model, we used adjusted hazard ratio (HR) estimates for 1-year mortality from the multivariate model. The adjusted HRs were converted into integer weights according to our previously established criteriaThe distribution of death according to time is described in eTable 2 of the t test from the 50 bootstrap samples. Kaplan-Meier curves for survival were computed for risk groups defined by the different indices.Each risk model was validated in an independent set of 367 patients . Overall, 605 patients (55%) were male, and 495 (45%) were female. eTable 4 in the 3 cells/μL), and high LDH level (>200 U/L) values met the predetermined significance level (P < .10) in univariate analyses to be considered in multivariate models. In the multivariate analysis, 9 comorbidities met the predetermined cutoffs (HR >1.2) for adjusted HRs to be assigned a score for the AML-CI , while an LDH value greater than 1000 U/L was assigned a score of 2.0 . Those 9 comorbidities constituted the new AML-CI.eTable 2 in the e AML-CI .  cytogenetic/molecular risk groups,P = .004) and AUC (P = .01)  . Age andP = .01) ; when boP < .001) and AUC of 0.76 (0.03) (P < .001) of the AML-CM were statistically significantly higher compared with those  of the augmented HCT-CI. The AML-CM also outperformed the Karnofsky performance status (KPS) . Visual representations of the higher C statistic and AUC values and hence better discriminative ability afforded by the AML-CM compared with age, KPS, ELN cytogenetic/molecular risk, the HCT-CI, the AML-CI, and the augmented HCT-CI can be found in the When age and cytogenetic/molecular risk groups were added to the augmented HCT-CI to form the AML-CM, this new composite model had a C statistic of 0.72 and AUC of 0.76 for 1-year mortality, and an AUC of 0.78 for 8-week mortality; all exceeded those of individual risk factors or models including performance status . For 1-yThe higher performance of the AML-CM persisted in comparison with KPS. The AML-CM provided better discrimination of mortality rates compared with all other risk factors, as illustrated in the Patients with AML have a relatively high early mortality rate of 37.5% at 8 weeksIn the current study, we have shown and validated that comorbidities have a significant impact on early and 1-year mortality among patients newly diagnosed with AML undergoing upfront therapies. In addition, we have demonstrated that an augmented HCT-CI performs better than either an AML-CI or the original HCT-CI in predicting early and late mortality. The HCT-CI has been in practice since 2005, making it quite familiar to and easily applicable by physicians. Most notably, when we incorporated comorbidities together with age and cytogenetic/molecular risks per the ELN classification into a composite model (AML-CM), this risk model performed better than each of the risk components alone . Just asNCT01929408). Finally, we did not incorporate regimen intensity, albeit predictive of mortality, in our model since this is the decision that we plan to improve based on the AML-CM scores.On the one hand, the inability to develop a comorbidity index specific to AML that outperforms the original HCT-CI matches our experience in another setting, the prediction of graft-vs-host disease after allogeneic HCT.NCT01929408) seeks to prospectively validate the AML-CM in patients with newly diagnosed AML. The AML-CM was designed to primarily predict survival at 1 year. The 1-year end point is intuitively appealing, and incorporates both treatment-related mortality and inherent resistance to antileukemic therapy. In contrast, an earlier end point may have lent undue significance to the former and a significantly later end point undue significance to the latter consideration. Nevertheless, the AML-CM strongly predicted earlier mortality at 8 weeks after initial therapy. Finally, although many patients receiving therapies of differing intensities were included, the intensity of therapy was not randomly assigned. We arbitrarily categorized those regimens based on feedback from all study collaborators into 3 levels of intensity. However, we did not incorporate regimen intensity, albeit predictive of mortality, in our model since what constitutes high-, intermediate-, and low-intensity therapy is arguable, and we are in the process of developing objective means to assess regimen intensity and to formally assess the benefits of regimens, thus defined, according to AML-CM scores.Limitations of the current study include the retrospective nature of data collection, potentially leading to failure to capture important data that might not have been recorded. However, the frequent use of laboratory data, most of which are consistently available in databases, to define comorbidities likely reduced the possibility of missing comorbidities. Most notably, the collected data for components of the HCT-CI were almost complete. Moreover, missing data on a few other comorbidities or covariates did not exceed 10%, and we have no reason to believe that missing data from retrospective medical record review were systematically related to outcome or other predictive factors. We confirmed that missing vs nonmissing data were not associated with outcome in this study. Regarding reproducibility of comorbidity assessments, while we did not perform analyses on interrater (IRR) and test-retest reliabilities for the augmented HCT-CI, we used recently described methods for comorbidity evaluation that indicated an IRR greater than 0.90 by weighted κ statistics.subjective assessment of a patient’s fitness. The use of the AML-CM by community internists will allow objective identification of those older patients with AML, who have traditionally been offered palliative care but who might be better served by referral to receive either conventional or investigational AML therapy. Consequently, some patients with AML who currently may not be referred for specific treatment will be referred in the future, leading to improved survival of patients nationwide. Conversely, the AML-CM could identify patients so unlikely to benefit from AML therapy that they could be spared the various risks and toxic effects of such therapies. In the future, the AML-CM might also be used to identify which patients would benefit more from intensive chemotherapy and which will be better served by targeted, less-intensive therapies, and we are organizing a national effort to examine this question. Whether the AML-CM calculated at diagnosis of AML can be later used to decide on allogeneic HCT vs non-HCT therapies is currently being studied in an ongoing observational study (NCT01929408). The AML-CM may also allow for more objective comparisons of outcomes with different therapies or with the same treatment at different centers. Finally, the identification of patients with high comorbidity burden at diagnosis will provide impetus for interventions to reduce the effects of these comorbidities before or simultaneously with the administration of initial AML therapy.The previous limitations notwithstanding, our results have significant clinical and scientific applications. Currently, decisions about initial therapy for newly diagnosed AML are based largely on age and an often http://www.AMLCompositeModel.org).We have developed a novel AML composite model that allows us to balance the effect of age with the effects of the patients’ overall health, as assessed by comorbidities, and the aggressiveness of their AML, as assessed by cytogenetic/molecular features. The AML-CM outperforms age and KPS in predicting early and late mortality and hence could replace these conventional covariates when making treatment decisions or comparing trial results. This model could prove useful to the US Food and Drug Administration when monitoring clinical trials to ensure adequate representation of high-risk patients in these trials and, hence, generalizability of trial results to the whole AML population. To facilitate the future use of this model, we are constructing a web-based calculator (


The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC), and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC) entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome-wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus-associated diseases.Many viruses modulate calcium (Ca Excess Ca2+ is removed from the cytosol by ATPases such as plasma membrane (PM) Ca2+-ATPases (PMCA), which pumps Ca2+ out of the cell and into the extracellular matrix, and sarcoplasmic reticulum Ca2+-ATPases (SERCA), which pump Ca2+ from the cytosol into the ER [2+]c and/or cytosolic Ca2+ signaling can affect development and progression of many diseases, including heart disease, schizophrenia, bipolar disorder, and Alzheimer’s disease. Moreover, disruption of Ca2+ signaling has been linked to cancer initiation, progression, metastasis, invasion, and tumor-associated angiogenesis [2+]c has also been implicated in the proliferation of human hepatoma cells [2+ signaling regulators has been associated with the development of hepatocellular carcinoma (HCC) [2+ signaling and elevated [Ca2+]c has been observed in cells with replicating hepatitis B virus (HBV) and hepatitis C virus (HCV); these viruses are currently the most common causes of HCC. These observations provide support for the notion that viral modulation of cellular Ca2+ signaling pathways could directly influence the development of viral-associated diseases.Intracellular Can a cell . In non-o the ER . Deregulogenesis , 9. Altema cells , and modma (HCC) . InteresHepadnaviridae family, and its genome is a small, partially double-stranded DNA of 3.2 kb in length containing four overlapping open reading frames that encode just seven proteins c in some established cell lines c in normal hepatocytes has not been assessed. We previously demonstrated that HBx enhances extracellular Ca2+ influx and mitochondrial Ca2+ uptake in human hepatoblastoma HepG2 cells [2+ levels in normal hepatocytes [2+]c in cultured primary rat hepatocytes following induction of an IP3-linked Ca2+ response and that HBx elevation of [Ca2+]c stimulates HBV replication in normal hepatocytes. We used both targeted and transcriptome-wide approaches to compare expression levels of store-operated Ca2+ (SOC) channel components and other Ca2+ signaling factors in HBV-expressing and control primary hepatocytes. HBV had no effect on the transcript level of any SOC channel component or Ca2+-signaling regulator that was detected in our analyses. Instead, we directly link HBx elevation of [Ca2+]c and HBx stimulation of HBV replication in hepatocytes to increased Ca2+ entry into hepatocytes through mitochondrial regulation of SOC entry (SOCE). Cumulatively, the results of our studies suggest that HBV (and HBx) does not affect the expression level or activity of SOC channel components but does increase, and likely prolong, Ca2+ entry through SOC channels to stimulate HBV replication in normal hepatocytes. The results from this study contribute to our current understanding of mechanisms that regulate HBV replication in normal hepatocytes and HBx effects that could influence the development of HBV-associated HCC. These studies may also provide insights into mechanisms that other viruses might employ to alter [Ca2+]c and Ca2+ signaling to enhance their own replication.The results of previously published studies have demonstrated that HBx expression causes an increase in the basal  bovine serum albumin [BSA], 200 μM BSP, pH 7.4). Hepatocytes were loaded with 5 μM Fura-4F-AM in HBSS supplemented with 0.02% pluronic acid for 40 min in 37°C shaker at 100 rpm, washed twice with HBSS, and then imaged using an Olympus 1X71 inverted microscope equipped with a 20X objective used for Fura imaging and a cooled charged-coupled-device camera. HBx-expressing and control cells were identified by dsRED expression and selected for [Ca2+]c measurements. To initiate Ca2+ release from the ER, 100 μM ATP or 100 nM Vp was added to the imaging buffer. Cytosolic Ca2+ signals were monitored by alternately exciting the dye at 340 nm and 380 nm and collecting emission at 510 nm with MetaFluor fluorescence ratio imaging software . Fura-4F fluorescence images were recorded every 2 seconds, and the change in [Ca2+]c was calculated as the change in the Fura-4F 340/380 nm ratio. Following each experiment, auto-fluorescence was estimated by treating cells with 10 μM ionomycin and 20 mM MnCl2 in Ca2+-free ECM to quench the Fura signal. Any remaining signals at 340 or 380 nm were subtracted prior to calculating the 340/380 nm ratio.Hepatocytes were plated on collagen-coated 15 mm glass coverslips immediately following isolation. 24 hours post plating, hepatocytes were transfected with 1 μg control vector (pGEM or pGEMHBV*7 or pcDNA3.1(-)) or 1 μg HBx expression vector (pGEMHBV or pcDNAHBx) and 0.2 μg dsRED. 24 hours post transfection, cells were washed twice with HEPES-buffered balanced salt solution (HBSS) . Statistical significance was determined using a two-tailed, nonparametric Student t test. A P value of ≤ 0.05 is considered statistically significant.For all CaPower SYBR Green master mix (Invitrogen), according to manufacturer’s instructions. Fold differences in the expression of SOC channel and mitochondrial Ca2+ signaling component mRNAs were calculated by the delta-delta Ct method c in cultured primary rat hepatocytes. Intracellular Ca2+-signaling studies are typically performed by the addition of a Ca2+-inducing agonist and subsequent measurement of the resultant intracellular Ca2+ response in cells that are loaded with a Ca2+ indicator; we utilized this technique to compare the intracellular Ca2+ responses of control and HBV-expressing hepatocytes. HBV has a very narrow host range and can only naturally infect primary human hepatocytes c in a single cell can be measured. We utilized ATP, a P2Y purinergic receptor agonist, to induce an IP3-linked cytosolic Ca2+ response in HBV-expressing and control hepatocytes , which binds to the vasopressin receptor V1 and induces an IP3-linked cytosolic Ca2+ response c was observed when HBx was expressed alone or in the context of the full HBV genome . The ER is naturally leaky, and the addition of TG causes a complete depletion of the ER Ca2+ store, thereby activating the SOC channel. Following ER store depletion, Ca2+ was added back to the imaging buffer; increased Fura-4F ratios reflect Ca2+ entering the cell through the open SOC channel c, similar to what we report here for HBV. Virus-mediated elevation of [Ca2+]c is often linked to activities of a viral regulatory protein, although for many viruses, the mechanisms that underlie regulation of [Ca2+]c are not fully defined c for other viruses that do not contain viroporins and that do not alter protein expression of SOC channel components.Modulation of Caes (HHV) , 7. Beca defined , 7. The  defined , 89, 90.activity . Alternaeristics . We also2+ in normal hepatocytes to altered feedback mechanisms that negatively regulate SOCE, specifically mitochondrial regulation of SOC channels. By blocking mitochondrial uptake of Ca2+, we prevented HBx from elevating cytosolic Ca2+ levels that were associated with activation of SOCE (2+ uptake seen in HBV- and HBx-expressing hepatocytes could be the result of HBx OMM localization and/or interaction with VDAC. Mitochondria normally take up cytosolic Ca2+ when the cytosolic Ca2+ level reaches 1 μM, which is 10 times the normal cytosolic concentration m in HBV-expressing hepatocytes. Our observed link of HBx elevation of cytosolic Ca2+ to mitochondrial-dependent processes is contradictory to a previous study conducted in HeLa and HepG2 cells where HBx elevation of cytosolic Ca2+ was associated with altered PMCA activity [2+]c out of the cell; when PMCA activity is altered or impaired, [Ca2+]c could accumulate in the cytosol c c . It is i [Ca2+]c . Moreove [Ca2+]c , 36. Ove2+ may also be linked to the development, progression, and/or maintenance of HBV-associated diseases. Hepatocytes are constantly exposed to various insults, toxins, and growth hormones that could initiate an IP3-linked Ca2+ response [2+]c is quickly corrected by shuttling this excess cytoplasmic Ca2+ out of the cell, through PMCA, or back into the ER, through SERCA. In HBV-infected hepatocytes, however, this regulation of Ca2+ levels and signals is altered, and HBV-infected hepatocytes are subjected to a more substantial and persistent increase in cytosolic Ca2+. Persistent elevation of cytosolic Ca2+ likely stimulates downstream Ca2+-dependent effector proteins and signaling pathways to create a cellular environment that is permissive to HBV replication, but consequently alters hepatocyte physiology, potentially influencing the development of HBV-associated diseases.In addition to regulating HBV replication, HBx elevation of cytosolic Caresponse –99. In n2+]c in normal hepatocytes to stimulate HBV replication; we have linked this HBx effect to mitochondrial regulation of [Ca2+]c. HBx is the only HBV regulatory protein and is required for HBV replication in human hepatocytes [2+]c could sensitize cells to signals that promote liver disease and/or HCC development. Importantly, HBx modulation of cytosolic Ca2+ is required for many of its effects in cells, indicating that HBx modulation of cytosolic Ca2+ is likely an upstream, initiating event that stimulates numerous downstream cellular functions [In summary, we have shown for the first time that HBV, via HBx, modulates SOCE to elevate [Caatocytes –22. HBx atocytes . HBV-relunctions , 29. Our