We postulated that if significant Ca2+ uptake into the matrix occurs from the lumen of the cristae, reduced expression of OPA1 would increase the access of Ca2+ to the transporters in the crista membrane and thus would enhance Ca2+ uptake. In intact H295R adrenocortical and HeLa cells cytosolic Ca2+ signals evoked with K+ and histamine, respectively, were transferred into the mitochondria. The rate and amplitude of mitochondrial [Ca2+] rise  were increased after knockdown of OPA1, as compared with cells transfected with control RNA or mitofusin1 siRNA. Ca2+ uptake was enhanced despite reduced mitochondrial membrane potential. In permeabilized cells the rate of Ca2+ uptake by depolarized mitochondria was also increased in OPA1-silenced cells. The participation of Na+/Ca2+ and Ca2+/H+ antiporters in this transport process is indicated by pharmacological data. Altogether, our observations reveal the significance of OPA1 in the control of mitochondrial Ca2+ metabolism.The dynamin-related GTPase protein OPA1, localized in the intermembrane space and tethered to the inner membrane of mitochondria, participates in the fusion of these organelles. Its mutation is the most prevalent cause of Autosomal Dominant Optic Atrophy. OPA1 controls the diameter of the junctions between the boundary part of the inner membrane and the membrane of cristae and reduces the diffusibility of cytochrome  Recent observations obtained in imaging and electron tomographic studies revealed a dynamically changing structure OPA1 gene is the most prevalent cause of the type 1 Autosomal Dominant Optic Atrophy The fusion of mitochondria is regulated by the transmembrane GTPase proteins mitofusin (Mfn) 1 and 2 and OPA1 a to e) of molecular weight, ranging from 94 to 86 kDa, can be found. Two long isoforms are anchored to the IMM and three soluble short forms are located in the IMS. These latter bands are the proteolytic products of the long forms OPA1  is a dynamin-related GTPase protein. Due to alternative splicing, its gene is transcribed into 8 mRNA isoforms Mfn1OPA1 gene brings about the fragmentation of the mitochondria OPA1 (or Mgm1) gene also evokes drastic desorganisation of the cristae OPA1 regulates the diameter of the crista junction. The tightness of junctions correlates with the oligomerization of the membrane-bound and the soluble forms in the IMS c reductase, F1F0 ATPase c oxidase c is found free in the IMS, while the major fraction can be found in the cristae OPA1 gene The role of the junction in the control of mitochondrial metabolism has been suggested by numerous observations. The majority of cytochrome 2+ signal that is rapidly transferred into the mitochondrial matrix. This sequestration of Ca2+ shapes the cytosolic Ca2+ signal and thereby modifies all the Ca2+ transport processes. The mitochondrial Ca2+ signal enhances the reduction of mitochondrial pyridine nucleotides and thus potentiates ATP formation , the driving force of the transport is the mitochondrial membrane potential   in the crista membrane with immunoelectron microscopy 2+ transport is indicated within the cristae 2+ uptake, suggesting the significance of the crista membrane in Ca2+ uptake. Our results imply that OPA1 may be a target of factors controlling mitochondrial Ca2+ metabolism.Calcium mobilising agonists generate cytosolic Ca2+ uptake we depressed its expression with siRNA. Since silencing of OPA1 has been known to evoke fragmentation of the mitochondria  RNA, the morphology of mitochondria was compared in the three groups. As shown in OPA1 and Mfn1-silenced cells. In H295R cells the median value of the length of single mitochondria diminished from 2.30 µm in control RNA-treated cells to 1.08 and 0.98 µm in cells exposed to Mfn1 and OPA1 siRNA, respectively  in intact cells the FRET-based, mitochondrially targeted, Cameleon-derived fluorescent protein 4mt-D1-cpV d for Ca2+ was about 10 µM and 85 nM, respectively. . The FRET ratio of the fluorescent proteins was insensitive to pH in the 7.6–8.2 range (data not shown). Fura-2 and 4mt-D2-cpV proved to be the appropriate sensors in H295R cells whereas Fura-FF and 4mt-D1-pcV were applied in HeLa cells.For monitoring cytosolic Ca2+ signal, elicited with 25 mM K+, into the mitochondrial matrix. Whereas the cytosolic signals were almost identical in the Mfn1 and OPA1 siRNA-treated cells, the FRET ratio of 4mt-D2-pcV, reflecting [Ca2+]m, showed a significant increase in the latter group  was related to peak [Ca2+]c, the normalized mitochondrial response was again significantly higher in the OPA1-silenced cells (p = 0.0015). The rate of mitochondrial Ca2+ uptake (indicated by the slope of the FRET ratio increase) showed a considerable variance, however, when it was analyzed in function of [Ca2+]c, it was found to be significantly higher in the OPA1-silenced than in the Mfn1-silenced cells (p = 0.039) In intact H295R cells we examined the transfer of cytosolic Ca= 0.039) . >1.25, the [Ca2+]m peak, Δ[Ca2+]m normalized to [Ca2+]c peak and the slope of [Ca2+]m increase normalized to peak [Ca2+]c were all significantly greater in OPA1-silenced than in Mfn1-silenced cells  . While transfection with various RNAs did not influence the cytosolic Caresponse . When st, resp.) .2+ uptake can be attributed to increased mitochondrial membrane potential (Ψm), increased activity of the Ca2+ transporting system as well as enhanced access of Ca2+ to the transporter. Assessment of Ψm with tetramethyl rhodamine methylester (TMRM) in H295R cells revealed that mitochondria were depolarized in OPA1 siRNA-transfected cells as opposed to the effect of Mfn1 siRNA  also in HeLa cells (p = 0.0001). The reduction of Ψm has been confirmed with JC-1   as compared with the effect of control RNA and amounted nearly to the double of that measured in Mfn1 silenced cells . Moreover, OPA1-knockdown augmented Ca2+ uptake rate when compared to control RNA treatment . Its high Ca2+ uptake by MCU (in polarized mitochondria) and by the H+/Ca2+ antiporter (Letm1) is inhibited by Ruthenium Red. In permeabilized, OPA1-silenced H295R cells the drug (15 µM) strongly inhibited Ca2+ uptake rate (p = 0.00016). CGP-37157 (25 µM), an inhibitor of the mitochondrial Na+/Ca2+ exchanger exerted a similar effect (p = 0.00016). No inhibition was observed with cyclosporin A (10 µM), an inhibitor of the mitochondrial permeability transition pore (mPTP) (2+ uptake rate to one fifth of the control (p = 0.00001) in permeabilized HeLa cells transfected with OPA1 siRNA. CGP-37157 and cyclosporin A failed to exert any effect on Ca2+ uptake  . Rutheni+ uptake .2+ accumulation occurs predominantly through the recently identified MCU protein 2+ channel +/Ca2+ exchanger (NCLX) and/or the Ca2+/H+ exchanger (Letm1) may be responsible for mitochondrial Ca2+ accumulation +/Ca2+ exchanger predominantly in the crista membrane 2+ uptake occurs exclusively on the inner boundary membrane as suggested in the schemes of several recently published reviews  Had the density or affinity of the transporter increased, enhanced Ca2+ accumulation could be expected even in the presence of lower [Ca2+]c but this was not the case in HeLa cells. Therefore it can be considered that, when [Ca2+]c is high, Ca2+ supply of the transporters located in the crista membrane is a limiting factor. Enhanced access of Ca2+ to these transporters in OPA1-silenced cells then augments the efficiency of the Ca2+ uptake process. Nevertheless, the possibility should be kept in mind that if ablation of OPA1 evokes the relocation of the transporter molecules from the crista into the boundary membrane, mitochondrial Ca2+ metabolism would alter similarly to the present observations. Unfortunately, this is difficult to prove or disprove at this stage.Ca2+ uptake in polarized mitochondria is the MCU 2+ transporting capacity of the supposedly electrogenic H+/Ca2+ antiporter 2+ uptake in our cells argues against any role of the rapid mode of Ca2+ uptake 2+ uptake in intact, OPA1-silenced cells is compatible with the presence of MCU in the crista membrane.Which transport mechanism is located in the crista membrane? The predominant mechanism of CaOPA1 siRNA-transfected, permeabilized cells after depolarizing the mitochondria. MCU is inactive in depolarized mitochondria 2+ uptake by depolarized mitochondria in permeabilized HeLa cells was reduced by Ruthenium Red. The Ca2+/H+ exchanger Letm1 is expressed in HeLa cells and is inhibited by Ruthenium Red 2+ uptake was not influenced by CGP-37157, an inhibitor of the Na+/Ca2+ antiporter. In the excitable cell type H295R Ruthenium Red almost completely abolished Ca2+ uptake and CGP-37157 also exerted a strong inhibition 2+/H+ and Na+/Ca2+ exchanger in H295R cells and the former one in HeLa cells participate in the enhanced Ca2+ uptake after knockdown of OPA1. In this respect it is worthwhile to recall that both antiporters are present in the adrenal cortex 2+ uptake through the crista membrane.In a study on murine retinal ganglion cells, transiently stimulated with K+, the subsequently added protonophore induced bigger cytosolic Ca2+ signals in OPA1-silenced than in control cells 2+ release and elimination of cytosolic Ca2+ by Ca.ATPases, can be attributed to changes in mitochondrial Ca2+ accumulation. However, in lack of appropriate control with fragmented mitochondria, the role of changes in various transports processes  should also be considered. Our direct measurements of mitochondrial Ca2+ metabolism not only demonstrated the increased rate of Ca2+ uptake in OPA1-silenced cells but also excluded the role of mitochondrial membrane potential in this action of the protein and suggested the presence of Ruthenium Red-sensitive transport mechanisms in the crista membrane.The transport mechanism was further analyzed in 2+ uptake rate and augments the mitochondrial Ca2+ signal. These results unambiguously demonstrate that OPA1 restrains mitochondrial Ca2+ uptake. On the basis of published data we attribute the uptake attenuating effect of OPA1 to decreased permeation of Ca2+ through the junction of the cristae. It follows that enhanced Ca2+ uptake in the absence of normal OPA1 function may be an aggravating component of OPA1-related diseases. Therefore OPA1 may be a target of factors modifying mitochondrial Ca2+ handling.Summarizing, the reduction of OPA1 expression results in enhanced mitochondrial Ca+, 2% UltroSer G, 100 U/ml penicillin and 100 µg/ml streptomycin. HeLa cells  were grown in DMEM containing 10% heat-inactivated FBS, 100 U/ml penicillin and 100 ug/ml streptomycin. Passage numbers 3–20 were used.H295R cells  were grown in DMEM/Ham's F12 (1∶1 v/v) containing 1% ITS4 H295R or 104 HeLa) were plated onto 24-mm diameter circular glass coverslips on day 1. For plasmid transfection we used 1 µg DNA (or 2 µg in case of H295R transfection with 4mt-D2-cpV) with transfection reagent (2 µl Lipofectamine 2000 for H295R or 2–3 µl FuGENE-HD for HeLa cells) in 1.1 ml OPTI-MEM medium. The transfection was performed on day 2 or 3. For silencing OPA1 a mixture of three siRNA species (1299003) was applied, for that of Mfn1 the product 5141600 and for control a non-silencing RNA with appropriate GC content (12935400 or 129305200), all from Invitrogen . Transfection took place on day 2, 40 pmol siRNA were added with 1 µl Lipofectamine RNAiMAX in 1.1 ml OPTI-MEM medium. When RNA was co-transfected with the plasmid coding for the Ca2+ sensitive protein, the protocol for plasmid transfection was applied. In experiments on intact H295R cells RNA transfection was repeated on day 3. The transfection reagents were purchased from Invitrogen, with the exception of FuGENE-HD . The experiments were conducted 56–76 hours after the first transfection.Cells . Insoluble fraction was removed with centrifugation. Protein concentration was measured with Bradford or BCA assay. The supernatant was completed with 1/3 volume reducing buffer . Samples were run on 8% SDS-PAGE and transferred onto nitrocellulose membrane (pore size: 0.45 µm). The membrane was incubated with 3% milk powder+0.1% Tween 20 in PBS to block nonspecific binding sites. Detection of OPA1 was performed with anti-OPA1 mouse monoclonal antibody  followed by incubation with anti-mouse immunoglobulin-horseradish-peroxidase conjugate (1∶2000) . Protein disulphide isomerase was used as loading control .102+ uptake and mitochondrial membrane potential were examined with confocal microscopy. Cells plated onto glass coverslips were placed on the stage of Zeiss LSM510 confocal laser scanning microscope equipped with a 40×/1.3 oil immersion objective (Plan-Neofluar Zeiss). Mt-inv-Pericam  was excited at 488 nm; emitted light was filtered using BP 500–550 nm emission filter. Rhod-2 (3 µM for 15 min) was excited at 543 nm; the emitted light was filtered using LP 560 filter. The optical slice was 5 µm, image acquisition frequency in the Ca2+ uptake experiments was 1-0.2 Hz. The experiments were performed at room temperature. The solutions were applied with a solenoid valve-equipped, gravity-driven superfusion system, terminating at ∼2 mm from the selected cells. Flow rate was ∼1 ml/min. Fluorescence intensity was normalized to the intensity measured before stimulation. The initial linear section of the normalized curves was regarded as rate of Ca2+ uptake. Uptake rate was expressed as (ΔF/F0)/Δt (Rhod-2) or ((ΔF−Fmin)/(F0−Fmin))/Δt (mt-inv-Pericam) where F/F0 is fluorescence intensity (F) related to that measured during the control period (F0) and Fmin is the fluorescence measured at saturating [Ca2+]c.The rate of mitochondrial Cam were followed with TMRE or TMRM. Cells preloaded with the dye were incubated in the presence of 25 nM dye. TMRE or TMRM was excited at 543 nm, emitted light was filtered using LP 560 emission filter. Alternatively, Ψm was examined with JC-1 (30-min loading with 1 µM at 37°C followed by 10-min incubation in the modified Krebs-Ringer solution without dye). JC-1 was monitored in “Multi Track” mode, excitation wavelengths of 488 nm and 543 nm were applied, emitted light was separated with a beam splitter cutting at 545 nm and emission filters BP 500–550 nm (green) and LP560 nm (red), resp., were used. The TMRE or TMRM fluorescence intensities and the ratios of red to green intensities in JC-1 experiments were normalized with that measured after depolarizing the mitochondria with FCCP.Changes in Ψc oxidase VIII target sequence. The length and circularity ((4π(area)/(perimeter)2) of mitochondria were analyzed with ImageJ 1.6.0, as suggested Mexican hat convolved duplicate – threshold – scale setting – analysis of particles.)For the morphological analysis of mitochondria the cells were transfected with GFP fused with human cytochrome 2+] was monitored with the appropriately targeted Ca2+ sensitive fluorescent proteins 4mt-D1-cpV  or 4mt-D2-cpV. In order to prepare 4mt-D2-cpV we cut out the 4-mt domain from 4mt-D1-cpV cloned in pcDNA3.1 with HindIII and inserted it in site of the 2-mt domain of 2mt-D2-cpV  also cloned in pcDNA3.1.Mitochondrial [Ca2+] was estimated with Fura-2 or Fura-FF, excited with 340 and 380 nm and using the above emission splitting system. Images were acquired every 5 seconds with the MetaFluor software (Molecular Devices) and MetaMorph was used for data analysis. FRET and Fura ratios were calculated after background subtraction; ratio of raw FRET acceptor (Venus) and donor (Cerulean) fluorescence or that of 340 nm/380 nm were used, respectively. Ratios were normalized to the control period (R0). Mitochondrial Ca2+ uptake rate was expressed as (ΔR/R0)/Δt. Measurements were performed at 31°C.Fluorescence intensity measurements were performed on an inverted microscope  equipped with a 40×1.4 oil immersion objective  and a Cascade II camera (Photometrics). Excitation wavelengths were set by a random access monochromator connected to a xenon arc lamp . For ratiometric FRET measurements of mitochondrially targeted D1 or D2-cpV excitation wavelength of 430 nm was selected along with a Dual-View emission splitting system  enabling the acquisition of simultaneous donor and raw FRET emissions. Alternately cytosolic  and [Mg2+] of the cytosol-like media, EGTA, HEDTA, CaCl2, MgCl2 and ADP were used as calculated by the Chelator software 2+] of the applied solutions was checked with a Ca2+ selective electrode . Calculated [Mg2+] was 0.5 mM.Incubation of intact cells was performed in a modified Krebs-Ringer solution containing 3.6 mM Kt-test (with or without Welch correction), one-way ANOVA, Tukey's or unequal N HSD post hoc tests or Kruskal-Wallis test were used, as appropriate. Data were analyzed with Statistica 9.Means + S.E.M. are shown. All the experiments were performed on at least two different cell passages. For estimating significance of differences, Student's unpaired Figure S1Morphology of the mitochondria of RNA-transfected H295R cells. The cells were transfected with control RNA (A), Mfn1 siRNA (B) or OPA1 siRNA (C) on the day following plating (day 2) and with mitochondrially targeted GFP on day 3. Confocal microscopy was performed on day 4. The framed areas are shown in the right-hand column; zoom: 4×. Optical slice thickness was 1 µm. Bars, 10 µm.(TIF)Click here for additional data file.Figure S2Morphology of the mitochondria of RNA-transfected HeLa cells. The cells were transfected with control RNA (A), Mfn1 siRNA (B) or OPA1 siRNA (C) on the day following plating (day 2) and with mitochondrially targeted GFP on day 3. Confocal microscopy was performed on day 5. The framed areas are shown in the right-hand column; zoom: 4×. Optical slice thickness was 1 µm. Bars, 10 µm.(TIF)Click here for additional data file.Figure S3Morphometry of the mitochondria of RNA-transfected cells. For the experimental protocol see the legend of A and B, resp.) and in HeLa cells . Analysis was performed with ImageJ 1.6.0, as suggested (TIF)Click here for additional data file.Figure S4Mitochondrial membrane potential of RNA-transfected HeLa cells. Transfection with control RNA, Mfn1 or OPA1 siRNA was performed on the day following plating. Three days later Ψm was estimated applying TMRM in H295R cells and TMRE or JC-1 in HeLa cells, respectively. TMRM and TMRE fluorescence or JC-1 ratio (red/green) over the mitochondrial region were normalized to that measured after depolarisation with FCCP. Means + SEM are shown, the number of observations is shown within the columns.(TIF)Click here for additional data file.Figure S52+ transport mechanism in depolarized mitochondria.Pharmacological characterization of the Ca H295R cells (A) or HeLa cells (B) were transfected with OPA1 siRNA 1 day after plating (day 2). On day 5 the cells were loaded with Rhod-2 AM, permeabilized and superfused with a cytosol-like medium. Ψm was dissipated , 25 µM CGP-37157 (CG) and 10 µM cyclosporine A (Cy), added alone or in combination, were present from the beginning of permeabilization. The means + SEM of the slopes of the initial increase in normalized Rhod-2 fluorescence are shown. The number of observations is shown within the columns. For the significance of differences see (TIF)Click here for additional data file.Table S1Significance of differences for the experiments shown in  C: control, RR: Ruthenium Red, CG: CGP-37157, Cy: cyclosporine A.(DOC)Click here for additional data file.


In unfractionated leukemic cells, submicromolar concentrations of JQ1 induced major growth-inhibitory effects (IC50 0.05-0.5 μM) in most samples, including cells derived from relapsed or refractory patients. In addition, JQ1 was found to induce apoptosis in CD34+/CD38− and CD34+/CD38+ stem- and progenitor cells in all donors examined as evidenced by combined surface/Annexin-V staining. Moreover, we were able to show that JQ1 synergizes with ARA-C in inducing growth inhibition in AML cells. Together, the BRD4-targeting drug JQ1 exerts major anti-leukemic effects in a broad range of human AML subtypes, including relapsed and refractory patients and all relevant stem- and progenitor cell compartments, including CD34+/CD38− and CD34+/CD38+ AML cells. These results characterize BRD4-inhibition as a promising new therapeutic approach in AML which should be further investigated in clinical trials.Acute myeloid leukemia (AML) is a life-threatening stem cell disease characterized by uncontrolled proliferation and accumulation of myeloblasts. Using an advanced RNAi screen-approach in an AML mouse model we have recently identified the epigenetic ‘reader’ BRD4 as a promising target in AML. In the current study, we asked whether inhibition of BRD4 by a small-molecule inhibitor, JQ1, leads to growth-inhibition and apoptosis in primary human AML stem- and progenitor cells. Primary cell samples were obtained from 37 patients with freshly diagnosed AML (n=23) or refractory AML (n=14). BRD4 was found to be expressed at the mRNA and protein level in unfractionated AML cells as well as in highly enriched CD34 Acute myeloid leukemia (AML) is a stem cell-derived hematopoietic malignancy characterized by uncontrolled proliferation and accumulation of myeloblasts in the bone marrow (BM), blood, and other organs. The clinical course and prognosis in AML vary, depending on age, the biology and category of the disease, cytogenetic features and number and types of deregulated genes -6. In a +/CD38− fraction of the leukemic clone [+/CD38+ and the CD34+/CD38− fraction of AML cells, or even in CD34− subfractions of the clone [One key event in oncogenic transformation in AML is the corruption of myeloid cell-fate programs resulting in the generation of aberrantly self-renewing cells, the so-called leukemic stem cells (LSC), which maintain and propagate the disease and are often resistant to conventional chemotherapy -16. Notaic clone . Other she clone ,23. Clinhe clone -32. Notahe clone -32.in vivo RNAi technologies. Through this approach we were able to identify the epigenetic ‘reader’ Bromodomain-containing 4 Protein (BRD4) as a new potential target in AML [In an effort to systematically probe genes involved in chromatin regulation as potential therapeutic targets, we have recently developed an unbiased screen approach, combining AML mouse models and new t in AML . Inhibitt in AML .In the present study, we extended these analyses to various subtypes of AML as well as to AML LSC. The specific aim of our study was to evaluate BRD4-inhibition as a potential therapeutic approach to target and eliminate LSC in AML. To address this question, we analyzed the effects of JQ1 on primary neoplastic stem- and progenitor cells obtained from patients with freshly diagnosed or refractory AML. In addition, we asked whether JQ1 would synergize with conventional cytostatic drugs to produce synergistic anti-leukemic effects in AML.+/CD38+ AML progenitor cells and CD34+/CD38− stem cells   without major differences when comparing freshly diagnosed AML patients with relapsed or refractory AML .Confirming our previous data  the BET ML Table . In normIn a next step, we examined the mechanism(s) of JQ1-induced growth inhibition in AML cells. In these experiments, JQ1 was found to induce apoptosis in primary AML cells in all samples tested, including cells derived from freshly diagnosed or relapsed patients. Induction of apoptosis was measured by light microscopy Figure  as well +/CD38+ as well as CD34+/CD38− stem and progenitor cells in all samples and all donors tested, including patients with relapsed or refractory AML, and without major differences when comparing FAB or WHO subtypes.In a next step, we examined the effects of JQ1 on survival of leukemic stem- and progenitor cells derived from our AML patients. As shown in Figure Since JQ1 and other (similar) BET bromodomain inhibitors are considered to be used in clinical trials in AML patients in combination with conventional cytostatic drugs like ARA-C, we asked whether JQ1 and ARA-C would produce cooperative or even synergistic growth-inhibitory effects on AML cells. For these experiments, the AML cell lines HL60 and KG1 were employed. As visible in Figure Based on their self-renewal and disease-propagating capacity, leukemic stem- and progenitor cells represent an attractive therapeutic target in AML -32. Notain vitro. However, the concentrations required to fully block growth of normal BM cells were higher as that required to block proliferation of AML blasts. This difference may well provide a “pharmacological window” sufficient to introduce JQ1 or similar BET bromodomain inhibitors in clinical trials. In line with this assumption, JQ1 was not found to act as a myelosuppressive agent when applied in vivo in various mouse models [Expression of BRD4 in AML stem- and progenitor cells was demonstrable by qPCR and by immunostaining. Both the CD34+/CD38- and the CD34+/CD38+ stem- and progenitor cells contained BRD4 mRNA as well as the BRD4 protein in all categories of AML, including refractory or relapsing disease. In addition, all AML cell lines examined were found to express BRD4. An interesting observation was that normal CD34+ stem cells also expressed BRD4, with similar expression levels compared to LSC. In addition, myeloid progenitor cells and even mature granulocytic cells were found to express BRD4 in the normal BM. These data suggest that targeting of BRD4 may result in a decreased production of normal BM cells. Indeed, we were able to show that JQ1 inhibits the growth of normal BM cells e models .BRD4 is a well known transcriptional regulator and chromatin modifier that plays a key role in cell cycle progression in normal and neoplastic cells -36. MoreWe have recently shown that the BRD4-blocking drug JQ1 induces growth inhibition and apoptosis in human and murine AML cells . In the As mentioned above, LSC are a major operational target of drug therapy -32,39. IMany of the novel anti-leukemic drugs used to treat refractory AML are applied in combination with other anti-AML agents -42. TherIn conclusion, we have identified BRD4 as a novel drug target expressed in AML cells including AML LSC, and that JQ1 is an effective agent capable of blocking growth of AML cells and LSC through BRD4 inhibition. These observations may have clinical implications, as BRD4 blockers like JQ1 and similar agents may soon be applied in clinical trials to treat AML. Whether these drugs will improve therapy in relapsed or resistant AML remains to be determined.A total number of 37 patients with AML  were examined. The median age was 59 years (range: 21-85 years). Diagnoses were established according to the proposal of the French-American-British (FAB) cooperative study group ,44 and t2 and 37°C.AML cell lines used in this study were HL60, U937, KG1, MV4-11, and MOLM-13 . Cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum  at 5% COTo confirm expression of BRD4 in leukemic cells, ICC and IHC were performed according to published protocols -48. ICC 2O2. Slides were incubated with anti-BRD4 antibody  at 4°C for 20 hours. Then, slides were washed and incubated with biotinylated anti-mouse IgG  for 30 minutes, washed, and then exposed to Vectastain ABC KIT for 30 minutes. 3-amino-9-ethylcarbazole  was used as chromogen. All slides were counterstained in Mayer´s Hemalaun.IHC was performed on sections prepared from paraffin-embedded, formalin-fixed BM biopsy specimens  using the indirect immunoperoxidase staining technique as reported -48. Prio4 cells/well) in RPMI 1640 medium (PAA laboratories) plus 10% FCS in the absence or presence of JQ1  at 37°C (5% CO2). After 48 hours, 0.5 μCi 3H-thymidine was added (16 hours). Cells were then harvested on filter membranes in a Filtermate 196 harvester . Filters were air-dried, and the bound radioactivity was measured in a ß-counter . All experiments were performed in triplicates. Proliferation was calculated as percent of medium control, and the inhibitory effects of JQ1 were expressed as IC50 values. In a separate set of experiments, AML cell lines  were incubated in various concentrations of JQ1, various concentrations of ARA-C, or a combination of both drugs at fixed ratio of drug-concentrations.Primary cells (MNC) were cultured in 96-well microtiter plates  (5-10 × 102) for 48 hours. Thereafter, cells were subjected to flow cytometry experiments following published protocols [Primary AML cells (n=9) were incubated in RPMI 1640 medium and 10% FCS in the absence or presence of various concentrations of JQ1  at 37°C  and Annexin-V-FITC  as described [To study apoptosis in stem- and progenitor cells, CD45escribed . Apoptos+/CD38− stem cells and CD34+/CD38+ progenitor cells were highly enriched by mAb and cell sorting essentially as described [In 15 patients with AML  and sorted cord blood CD34+/CD38+ and CD34+/CD34− cells pooled from 3 donors. RNA was isolated using RNeasy Mini-Kit . mRNA levels were quantified on a 7900HT Fast Real-Time PCR System from Applied Biosystems , using iTaq SYBR Green Supermix with ROX from Bio-Rad . ABL was employed as a reference gene. Primers used in this study were: hu BRD4-forward: 5'-GCCCGCAAGCTCCA GGATGT-3'; hu BRD4-reverse: 5'-CCTCAGGCTCGTCCGGCATC-3'; hu ABL-forward: 5'-TGTATGATTTTGTGGCCAGTGGAG-3'; and hu ABL-reverse: 5'-GC CTAAGACCCGGAGCTTTTCA-3'.PCR experiments were performed on RNA from AML cell lines, unfractionated AML cells, sorted CD34Differences in growth and apoptosis in drug-exposed cells were determined by appropriate statistical analysis, including the paired student´s t test. Results were considered to be significantly different when the p value was <0.05.


The results indicated that EGCG caused a dose-dependent increase in intracellular Ca2+ ([Ca2+]i). This [Ca2+]i increase was blocked by depleting intracellular Ca2+ stores with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin and cyclopiazonic acid. Furthermore, EGCG-stimulated increase in [Ca2+]i was abolished following treatment with a PLC inhibitor. However, EGCG inhibited high-voltage activated Ca2+ currents (IHVA) and NMDA-induced inward currents (INMDA). These data suggest that EGCG triggers a cascade of events: it activates phospholipase C (PLC), mobilizes intracellular Ca2+ stores, raises the cytosolic Ca2+ levels, and inhibits the VGCC and NMDA receptors-mediated Ca2+ influx through a process that remains to be determined.Green tea has been receiving considerable attention as a possible neuroprotective agent against neurodegenerative disease. Epigallocatechin-3-gallate (EGCG) is the major compound of green tea. Calcium signaling has profound effects on almost all aspects of neuronal function. Using digital calcium imaging and patch-clamp technique, we determined the effects of EGCG on Ca Green tea polyphenols are natural plant flavonoids and comprise many types of catechin. Among them, (–)-epigallocatechin gallate (EGCG) is the major polyphenol component and primarily responsible for the green tea effect. Previous studies on the biological activities of EGCG were mostly focused on its beneficial effects, including antioxidant ,2, antic2+) is a ubiquitous intracellular signal responsible for controlling numerous cellular processes, such as fertilization, proliferation, development, learning and memory i) in both non-excitable and excitable cells. EGCG reportedly attenuates the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced increase in [Ca2+]I in hippocampal neurons i increase by attenuating both ionotropic Ca2+ influx and Ca2+-induced Ca2+ release (CICR) in PC12 cells, but it did not affect matabotropic receptor-mediated Ca2+ release [2+]i in cultures of rat hippocampal neurons is partially responsible for the death of hippocampal neurons induced by EGCG [2+]i produces deleterious effects and results in cell death. Thus, it is essential for cells to carefully buffer intracellular calcium and to precisely regulate calcium entry [2+]i, which would be compensated for by the calcium-buffering mechanisms of cells. However, high-concentration EGCG produced excessive elevation of [Ca2+]i which was possible beyond the buffering capacity of neurons and led to a cascade of cellular pathological events i imaging technique to study the dynamic alteration of intracellular calcium mediated by EGCG in primary culture of rat hippocampal neurons. From the results, the effect of EGCG was significant at 10 μM , and reached a maximal level at a concentration of 100 μM (50 for potentiation effect was 11.6 ± 1.6 μM (fitted with Sigmaplot version 9.0 to the Hill equation). Calcium mobilization was initially detected 100 s following the application of 30 μM EGCG and rapidly reached a plateau in 236 ± 48 s. Repeated 30 μM EGCG stimulation produced equal response and this response was fully reversed after washout with ACSF. The response were detected in 59% (34 of 58 cells tested) of neurons, resulting in a 70.8 ± 11.0% increase over basal levels.In the first set of experiments, we employed the [Caf 100 μM . The EC52+ influx from the extracellular compartment or the release from intracellular Ca2+ stores on EGCG-induced [Ca2+]i elevation in hippocampal neurons, the standard extracellular solution was replaced by Ca2+-free solution. In the absence of extracellular Ca2+, 30 μM EGCG was able to induce a significant increase of basal [Ca2+]i , although the magnitude of elevation .To ascertain which intracellular signaling pathway is activated in EGCG-induced Ca n = 20, , whereas3 and 1,2-diacylglycerol (DAG) production. Subsequently, IP3 stimulates the Ca2+ release from endoplasmic reticulum (ER) stores; DAG leads to activation of protein kinase C (PKC). To test which pathway was involved in EGCG-mediated elevation of [Ca2+]i, primary cultured hippocampal neurons were pretreated with heparin , EGCG-evoked responses were attenuated to 39.6 ± 4.1% over basal levels . When cells were treated with PKC inhibitor GF109203X (2 μM), the stimulation effects of EGCG were not noticeably altered. The results suggested that the potentiation effect of EGCG was partially dependent on the activation of IP3. To determine whether coupling to the AC signaling pathway was affected by EGCG, cells treated with pCPT-cAMP, a membrane permeable cAMP analog, was examined. In contrast, pretreatment with 200 μM pCPT-cAMP for 3 min had no effect on the increase in EGCG-mediated intracellular calcium levels . Meanwhile, H-89, a permeable PKA inhibitor, did not alter EGCG-stimulated elevation . These data suggested that EGCG mediated calcium signaling was not dependent on PKA pathway, but a PLC dependent pathway.Activation of PLC is known to stimulated PI hydrolysis, IP2+ influx from the extracellular compartment contributed to the EGCG induced [Ca2+]i elevation, the exact pathway involved in this regulation is unclear. To address this issue, we first employed verapamil to block voltage-gated calcium channels (VGCC). As shown in n = 21, P < 0.05).Although we demonstrated that Ca2+]i. The maximal increases in [Ca2+]i over basal levels were 51.7 ± 7.2% . However, 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a selective AMPA/kainite receptor competitive antagonist, did not produce any inhibition on EGCG induced elevation of free calcium in the neurons  in this process was checked. As depicted in  neurons . This re2+]i elevation, the whole cell patch-clamp technique was employed to study the effects of EGCG on high-voltage-activated calcium currents (IHVA) and NMDA-induced inward currents (INMDA). IHVA were elicited by a depolarization from holding potential of −80 mV to +10 mV. EGCG 30 μM caused a reduction in IHVA from 63.2 ± 5.9 pA.pF−1 to 46.7 ± 5.3 pA.pF−1. IHVA decreased by EGCG was irreversible after EGCG was washed out (2+ chelator BAPTA (15 mM), which is sufficient to maintain [Ca2+]i at a nanomolar level. As shown in n = 8, P < 0.05 vs. EGCG alone). These results revealed that the inhibition induced by EGCG was Ca2+-dependent.To further determine the roles of VGCC and NMDA receptor in the EGCG induced i. is central to multiple signal transduction pathways to accomplish a variety of biological functions. Our result is in agreement with a previous report that EGCG could cause a [Ca2+]i elevation in a dose-dependent way [2+]i mobilization: an intracellular release through plasma membrane channels and a internal reservoir in the ER and mitochondria [2+]i in the absence of extracellular Ca2+ and was abolished in cells that were depleted of intracellular calcium stores by thapsigargin and CPA, which suggested that the mobilization of Ca2+ in the ER was necessary and sufficient for this process. However, inhibition of EGCG-induced [Ca2+]i increase by removal of extracellular Ca2+ recovered again after reperfusion with Ca2+-containing medium. These results suggest that Ca2+ influx is partly involved in the EGCG induced calcium elevation. In our present study, a large proportion of EGCG-induced elevation of [Ca2+]i was still apparent in the presence of VGCC and ROCC blockers, which suggested that these entry pathways did not represent the principal mode of elevation of [Ca2+]i. Verapamil and D-AP5 slightly reduced EGCG-induced increase in [Ca2+]i in hippocampal neurons. However, EGCG inhibited IHVA and INMDA, suggesting that the EGCG-induced Ca2+ influx occurred mainly though other pathways. Capacitative calcium entry (CCE) via store-operated calcium channel is considered to be the major mechanism for influx of Ca2+ in a nonexcitable cell system. It is generally believed that CCE operation is nonexistent in neurons, although there were some disagreements [2+ influx.[Cadent way . Two majchondria . Our datreements . Althoug2+]i would result in intracellular calcium overload, which triggers a cascade of events leading to cell death. Calcium-buffering mechanism is essential for cells to reduce [Ca2+]i. One target of intracellular calcium is voltage-dependent and ligand-gated ion channels i. A clear understanding of the signal transduction mechanisms is a key to the evaluation of action of EGCG, either as a neuroprotective agent or neurotoxic agent [3 receptors with heparin or depletion of intracellular calcium store with thapsigargin and CPA disrupted the EGCG-induced increase in [Ca2+]i. These data indicated that EGCG stimulated [Ca2+]i via PLC-IP3 pathway in hippocampal neurons.EGCG is known to bind to several proteins and affects the activity of enzymes and receptors. It has been reported to act through phosphoinositide 3-kinase (PI 3-kinase), Akt/protein kinase B (Akt/PKB), tyrosine kinases, protein kinase C (PKC), and mitogen activated protein kinase (MAP kinase) signaling cascades . Inhibitic agent . In our EGCG, U-73122,U-73343, p-CPT-cAMP, CPA, cyclopiazonic acid, NMDA, H-89, DL-2-amino-5-phos-phonovaleric acid (AP-5), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), heparin, GF109203X, verapamil, and thapsigargin were purchased from Sigma . Fura-2/AM was obtained from Biotium . DMEM/F12 and B27 supplement were obtained from Gibco Invitrogen Corporation . Other general agents were available commercially. Other agents were purchased from commercial suppliers. Agents were prepared as stock solutions with sterile water except difedipine, U-73122, U-73343 and Fura-2/AM, which were dissolved in dimethylsulfoxide (DMSO) and stored at −20 °C. They were diluted to the final concentrations before application. The final concentration of DMSO was <0.05%. No detectable effect of the vehicles was found in our experiments.National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experiments conformed to named local guidelines on the ethical use of animals and all efforts were exerted to minimize the number of animals used and their suffering. Neurons were isolated as previously described with some modification i. The intracellular free calcium concentration is presented as the ratio of the fluorescence signals obtained (340/380 nm). All experiments were repeated at least three times using different batches of cells.Digital calcium imaging was performed as described by Ming et al. . Hippoca2 2.0, Na2ATP 5.0, egtazic acid 11, HEPES 10, TEA-Cl 20, creatine phosphate 20, and the pH was adjusted to 7.2 with CsOH. The extracellular solution composed of (in mM) NaCl 110, KCl 5.0, CaCl2 5.0, MgCl2 1.0, HEPES 10, Glucose 11, 4-AP 5.0, TEA-Cl 25, TTX 0.5 × 10−3 and the pH was adjusted to 7.4 with NaOH. NMDA currents were recorded using electrodes filled with (concentration in mM) cesium gluconate 135, CsCl5, CaCl2 5.0, MgCl2 1.0, HEPES 10, Glucose 11, pH 7.2, with Tris base, and the bath solution contained (in mM) NaCl 140, KCl 5, CaCl2 2.5, HEPES 10, Glucose 10, TTX 0.5 × 10−3, pH 7.4 with Tris base. 100 μM NMDA was applied to cells for 15 s using a modified Y-tube. Drug actions were measured only after steady-state conditions reached, which were judged by the amplitudes and time courses of currents remaining constant.The procedure for whole cell patch-clamp recording was described in our previous study . Current2+]i elevation over the basal were represented as the difference between baseline level [(F340/F380)B] and the peak amplitude [(F340/F380)S] response to the stimulation, which was shown as [(F340/F380)S-F340/F380)B]/(F340/F380)B. Data are presented as mean ± SEM. Data were analyzed using SPSS 10.0 software. Student’s t-test or one-way ANOVA was used to test for significance. Differences were considered significant at P < 0.05.The amplitudes of i and inhibited IHVA and INMDA in cultured hippocampal neurons remain a matter of conjecture, our results may help to understand the neurotoxicity and officinal value of EGCG.Results presented here suggested that EGCG triggered a cascade of events: it activated phospholipase C, mobilized intracellular calcium stores, raised the cytosolic Ca


Numerous factors impact on the prognosis of acute myeloid leukemia (AML), among which molecular genetic abnormalities are developed increasingly, however, accurate prediction for newly diagnosed AML patients remains unsatisfied. For further improving the prognosis evaluation system, we investigated the transcripts levels of PDCD7, FIS1, FAM3A, CA6, APP, KLRF1, ATCAY, GGT5 and Ang2 in 97 AML patients and 30 non-malignant controls, and validated using the published microarray data from 225 cytogenetically normal AML (CN-AML) patients treated according to the German AMLCG-1999 protocol. Real-time quantitative polymerase chain reaction and western blot were carried out, and clinical data were collected and analyzed. High Ang2 and FIS1 expression discriminated the CR rate of AML patients . In CN-AML, patients with high FIS1 expression were more likely to be resistant to two courses of induction (P = 0.035). Overall survival (OS) and relapse-free survival (RFS) were shorter in CN-AML patients with high PDCD7 expression , and PDCD7 was revealed to be an independent risk factor for OS in CN-AML (P = 0.004). In the analysis of published data from 225 CN-AML patients, PDCD7 remained independently predicting OS in CN-AML (P = 0.039). As a conclusion, Ang2 and FIS1 seem related to decreased CR rate of AML patients, and PDCD7 is associated with shorter OS and RFS in CN-AML. Hence, PDCD7, Ang2 and FIS1 may indicate a more aggressive form and poor prognosis of AML. Acute myeloid leukemia (AML) is a clonal hematopoietic stem cell malignancy with highly heterogeneity. The prognostic prediction for AML had been improved tremendously in the past decades, however, accurate risk-stratification at diagnosis remained difficult. Meanwhile, molecular mechanisms in the etiology and progression of AML are still vague, and AML genomes is expected to lead to identification of even more prognostic markers Twenty-two molecular targets, which are potentially associated with complete remission (CR) durations of AML, has been selected based on our previous gene-chip study Angiogenesis family play an important role in the growth and viability of myelogenous malignancies, and emerging data suggest a crucial involvement of the family member Angiopoietin-2 (Ang2) in this process. However, the role Ang2 played in AML remained controversial, such as the studies of Hou This study is aimed to investigate the expression status of the nine designated molecular markers including GGT5, FAM3A, PDCD7, FIS1, CA6, ATCAY, KLRF1, APP and Ang2, and their clinical relevance and prognostic significance in AML.Human samples left over from clinical examinations were collected, and each patients were informed consent in written form for their samples to be stored and used for research purposes in advance, we didn't take any samples from patients specifically for this study. All patients data were collected and analyzed anonymously. This study was retrospectively authorized by Nanfang Hospital Ethics Committee (2012-178).de novo (n = 93) or secondary AML (n = 4). All patients were diagnosed between September 2005 and July 2010 in Nanfang Hospital, Guangzhou, China. Diagnosis were based on the French-American-British (FAB) classification and World Health Orgnization (WHO) criteria th May 2013. The clinical features of AML patients are listed in Diagnostic bone marrow mononuclear cells were analyzed from 97 AML patients (age: 35 (13–65) years) with Bone marrow samples from 16 patients with iron deficiency anemia (IDA) and 14 patients with idiopathic thrombocytopenic purpura (ITP) were collected as control.Total cellular RNA was extracted using Ficoll-Hypaque gradient  and reversely transcribed to cDNA by PrimeScript™RT Reagent Kit .−ΔCt (ΔCt = Cttarget gene -Ctβ-actin)2RQ-PCR was performed using SYBR Premix Ex Taq™ according to the manufacturer's protocol on Mx3005P real-time PCR amplifier . Relative mRNA amount was calculated as Western blot were carried out following the procedure described in our previous study Data from 225 cytogenetically normal AML (CN-AML) patients treated according to the German AMLCG 1999 trial has been published on Gene Expression Omnibus . Datasets were mined for PDCD7 expression using Genebank accession number (AW953770) as published by National Center for Biotechnology Information (NCBI).We assigned AML patients into high and low expression groups by median relative mRNA transcript levels for each gene. Primary refractory AML patients included those resistant to two courses of induction For avoiding the interference from different cytogenetics, only CN-AML patients were included in survival analysis. CN-AML patients were divided into 3 groups by quartile of expression levels of target genes, and each patient was described by consolidation intensity. High intensity treatment included patients received ≥4 courses of MDAC/HDAC or HSCT, and low intensity included those received standard regimens from induction or <4 courses of MDAC/HDAC in consolidation .Expression data of PDCD7, FIS1, FAM3A, CA6, APP, KLRF1, ATCAY, GGT5 and Ang2 were analyzed in 97 AML patients and 30 non-malignant controls, as well as published data of PDCD7 expression from 225 CN-AML patients treated with a uniform protocol.AML patients had higher PDCD7 and lower KLRF1 expression levels than those of non-malignant controls . Primary refractory AML patients tended to express higher FIS1 transcripts than non-refractory patients (P = 0.059), for CN-AML, the tendency became statistically significant (P = 0.035), and was validated at protein level  2.564; 95% confidence interval (CI), 1.628–4.039; P<0.001; RFS: HR, 3.343; 95% CI, 1.412–7.916; P = 0.006; For further validate that our RQ-PCR results of PDCD7 on OS estimation were generalizable to AML patients irrespective of different chemotherapies, we analysed PDCD7 expression data from 225 CN-AML patients treated according to the German AMLCG-1999 protocol, and confirmed the prediction  encoding a 59 kDa protein, mainly works in U12-type minor spliceosome to interfere the splicing procession of mRNA precursors FIS1  homolog (S. cerevisiae)) has been well established in the etiology of neurodegenerative diseases The angiogenesis family member Ang2 was initially regarded as vascular remodeling antagonist of Ang1 KLRF1  expressed on the surface of most NK cells and a part of T cells In conclusion, PDCD7 predicted shorter OS and RFS in CN-AML, Ang2 and FIS1 related to CR response in AML. Thereby, PDCD7, Ang2 and FIS1 may indicate a more aggressive form and poor prognosis of AML.


Western blot analysis showed that GSK101 activation did not induce an increase in TRPV4 expression at the plasma membrane, but caused an immediate and sustained downregulation of TRPV4 on the plasma membrane in HeLa-TRPV4 cells. Patch clamp analysis also revealed an early partial desensitization of the channel which was Ca2+-independent. FRET analysis of TRPV4 subunit assembly demonstrated that the GSK101-induced TRPV4 channel activation/desensitization was not due to alterations in homotetrameric channel formation on the plasma membrane. It is concluded that GSK101 specifically activates TRPV4 channels, leading to a rapid partial desensitization and downregulation of the channel expression on the plasma membrane. TRPV4 subunit assembly appears to occur during trafficking from the ER/Golgi to the plasma membrane and is not altered by agonist stimulation.TRPV4  channels are activated by a wide range of stimuli, including hypotonic stress, non-noxious heat and mechanical stress and some small molecule agonists (e.g. phorbol ester 4α-PDD). GSK1016790A (GSK101) is a recently discovered specific small molecule agonist of TRPV4. Its effects on physical determinants of TRPV4 activity were evaluated in HeLa cells transiently transfected with TRPV4 (HeLa-TRPV4). GSK101 (10 nM) causes a TRPV4 specific Ca It is ubiquitously expressed in various tissues such as renal epithelia, lung epithelia, vascular endothelia, and nervous systems TRPV4 is a non-selective CaGSK101 (GSK1016790A) is a novel activator of TRPV4, which has been shown to be a more specific and potent activator (at nanomolarlevels) as compared to the traditional 4α-PDD2+-independent manner.In this study, we set out to investigate the relationship between GSK101-induced TRPV4 activation and its expression and subunit assembly at the plasma membrane as the physical determinants of TRPV4 activity. It was found that agonist stimulation did not alter the apparent subunit assembly within the plasma membrane, but it induced an early rapid downregulation of TRPV4 expression at the plasma membrane that was associated with a rapid desensitization of the TRPV4 channel in a CaRT-PCR using HeLa cell mRNA and primer pairs designed for TRPV channels showed that HeLa cells do not expressed TRPV4 channels at the mRNA level. Likewise, Western blot using anti-TRPV4 antibody also could not detect TRPV4 at the protein level in wild type HeLa cells (data not shown). Therefore, HeLa cells were used as an overexpression cell model when transiently transfected with a TRPV4 containing plasmid similar to that done previously in HEK and CHO cells 2+ influx was stimulated by GSK101 in a dose-dependent manner as shown in 2+ influx relationship could be fitted by asigmoidal dose-responsefunction, which yielded an EC50 of 3.3 nM for GSK101 stimulation. GSK101 at 10 nM displayed a near maximum stimulation and was, therefore, used as the preferred concentration to activate TRPV4 channels in this study.In HeLa-TRPV4 cells, Ca2+ concentration in HeLa cells transfected with mVenus-N1 vector, while HeLa cells transfected with TRPV4-mVenus responded rapidly to GSK101 with an immediate increase in intracellular Ca2+ concentration (2+ in HeLa TRPV4-mVenus cells averaged 267.5±44.3 nM (n = 6) while a significant change was not detected in HeLamVenus cells . This result further confirmed that GSK101 is a specific activator of TRPV4 that induces Ca2+ influx intoHeLa TRPV4-mVenus (HeLa-TRPV4) cells.In order to test the specificity of GSK101 on TRPV4 activation, HeLa cells transfected with mVenus-N1 vector were compared with HeLa cells transfected with TRPV4-mVenus. Only cells positively identified as expressing TRPV4-mVenus (via excitation at 488-nm wavelength) were chosen for the study. GSK101 did not cause an increase in intracellular Cantration . As show2+ in HeLa-TRPV4 cells was sustained over many minutes. While GSK101 induced an early peak Ca2+ influx, the intracellular Ca2+ levels only displayed a modest decay over time in the continued presence of the agonist, as shown by the average time course of the response in 2+ concentration in HeLa-TRPV4 reached its peak at 1.9±0.3 minutes (n = 8) and was maintained for over 30 minutes.The effect of GSK101 on intracellular Ca2+ concentration came from extracellular sources and/or intracellular stores, ruthenium red (RR), a TRPV channel inhibitor, and Ca2+-free media were utilized in calcium imaging experiments. As shown in 2+ influx. RR was more potent when added at an early stage of GSK101 activation (1–3 min post GSK101 activation). The inhibition by RR was reduced if added at a later time point (10 min post GSK101 activation). Addition of ionomycin, a Ca2+ionophore, on top of RR still caused a significant increase in intracellular Ca2+, indicating that fura 2 was not sequestered and fura 2 signaling was still active . Similarly, GSK101/Ca2+-free medium only modestly diminished GSK101-induced intracellular Ca2+ levels when the GSK101/Ca2+-free medium was applied at 10 min post GSK101activation. Ionomycin added on top of the Ca2+-free medium caused a moderate but transient increase in intracellular Ca2+ that was quickly diminished due to the depletion of Ca2+ in the environment , indicating this method is not ideal for studying pure plasma membrane proteins (data not shown). Instead, a sucrose-gradient based membrane protein extraction kit  showed very clean preparations of plasma membrane proteins without cytosolic contaminants, which was confirmed by non-detectable tubulin levels using anti-alpha tubulin antibody in Western blots .2+ influx, followed by channel endocytosis from the plasma membrane that may contribute to the channel desensitization.HeLa-TRPV4 cells were harvested and plasma membrane proteins purified at 0, 3, 10 and 30 min time points under GSK101 stimulation. The lysates were then probed with anti-TRPV4 antibody to test the channel expression. Wild type TRPV4 has a predicted molecular weight of 98 kDa; the TRPV4-mVenus then has a predicted molecular weight of 123 KD due to the 25 kDa tag. The lysates were also probed with anti-pan cadherin for loading control and with anti-alpha tubulin for detecting cytosolic contamination . DensitoRecently Shigematsu and coworkers have discovered that His-tagged rat TRPV4 forms a tetramer by using chemical cross-linking and Nanogold labeling in transmission electron microscopy HeLa cells were transiently co-transfected with TRPV4-mCerulean (donor) and TRPV4-mVenus (acceptor). The cells were fixed at four time points of GSK101 treatments, 0, 3, 10 and 30 min. A widely used FRET photo-bleaching protocol was used to bleach the TRPV4-mVenus with the 514 nm laser, the changes in fluorescence intensity in TRPV4-mCerulean were recorded and analyzed. 2+ influx in HeLa-TRPV4 cells, but not in control transfected HeLa-mVenus cells. To be more specific, it was observed that the early rise in intracellular Ca2+ levels reached a peak in 1.9 minutes and was sustained thereafter in HeLa-TRPV4 cells.This study evaluated the determinants of TRPV4 activity in a TRPV4 over-expression system, HeLa-TRPV4 cells. Our calcium imaging studies show that the small molecule agonist, GSK101, causes an early and rapid activation of TRPV4, leading to Ca2+ levels back towards basal values as typically observed for most Ca2+ signaling events since many TRP channels, including TRPV4 as discussed below, often desensitize following activation 2+ is not likely the result of Ca2+ influx only. The apparent sustained elevation in cytosolic Ca2+ levels is not an artifact relating to fura 2 being quenched or sequestered because ionomycin was still able to elicit a rise in intracellular Ca2+ levels when added at the end of the experiments. Hence, other factors controlling cytosolic Ca2+ homeostasis must be invoked in the HeLa-TRPV4 overexpression system, possibly induced calcium release from stores or impaired calcium extrusion from the cytoplasm. The reason for this late effect remains to be determined in future studies.It is of note that HeLa-TRPV4 cells did not return the GSK101-induced intracellular Ca2+-induced sensitization of the channel similar to that shown by others 2+-induced or Ca2+-calmodulin induced desensitization as noted for other channels, including TRP channels 2+-free extracellular solution showed an even larger transmembrane current with the similar activation pattern and outward rectification under GSK101 stimulation ; Ca2+ levels fell only minimally in the late stage (10 min activation). These studies again point to an early partial desensitization of TRPV4. Therefore, in the late stage of calcium imaging in HeLa-TRPV4 cells, the sustained intracellular Ca2+ concentrations above baseline is unlikely to be the direct effect of continued Ca2+ influx through TRPV4 channels alone, but rather the result of Ca2+ release from stores or diminished extrusion of Ca2+ from the cytosol via the plasma membrane or sarcoplasmic reticulum Ca2+ pumps, as heretofore described.The desensitization of TRPV4 following GSK101 stimulation is also supported by the calcium imaging data showing that the application of ruthenium red (3 µM) and CaWe directly evaluated the role of TRPV4 channel endocytosis from the plasma membrane as a determinant of TRPV4 activity. Ion channel trafficking to and from the plasma membrane is difficult to study, partly due to the difficulty in extracting pure plasma membrane proteins and the complex steps during vesicle trafficking. In this study, we utilized a sucrose gradient-based extraction method that has produced pure plasma membrane fractions in HeLa-TRPV4 cells. Anti-alpha tubulin antibody in Western blotting is a useful marker to check whether the plasma membrane fraction is contaminated with cytosolic proteins because cytoskeletal proteins are the most common contaminants during plasma membrane extraction. Acomparison of the traditional biotin-streptavidin system with the sucrose gradient-based extraction kit shows that the sucrose gradient-based extraction method yields a highly enriched, pure, plasma membrane fraction, while the biotin-streptavidin method produces a crude plasma membrane fraction with considerable contamination of cytoskeletal proteins. However, it is worth noting that sucrose gradient-based extraction has a lower yield in the final protein quantity than biotin-streptavidin system.2+ entry.The examples include insulin-induced TRPV2 channel insertion onto the plasma membrane in pancreatic β-cells 2+ serves as a negative modulator of TRPV4 membrane expression and initiates the endocytosis of TRPV4 from the plasma membrane, likely leading to reduced Ca2+ entry. Although the endocytosis mechanism is not known, the usual machinery for vesicle trafficking, such as Rab small GTPases, SNAREs and phosphoinisitides are likely to be involved and require further investigation Few studies have evaluated TRP channel trafficking and most have focused on translocation and insertion of TRP channel onto the plasma membrane during a stimulus. The hypothesis is that a stimulus causes channel exocytosis and insertion into the plasma membrane, resulting in an increased number of channels, leading to enhanced CaTo provide a more in depth assessment of the physical determinants of TRPV4 activation, we attempted to decipher the mechanism of GSK101 activation/desensitization of TRPV4 channels using a FRET analysis to evaluate the physical association among subunits of the TRPV4 homotetramer. Alterations in the physical associations between channel subunits are thought to play an important role in activation/modulation of TRP channels Sal I and Bam HI sites of mCerulean-N1 and mVenus-N1 vectors , so that the mCerulean or mVenus fusion protein was at the C-terminus of recombinant TRPV4 protein. To simplify the terminology in this work, TRPV4-mCerulean stands for mCerulean-N1-TRPV4 and TRPV4-mVenus stands for mVenus-N1-TRPV4. Both plasmids went through sequencing to verify the correct DNA sequences. The recombinant TRPV4-mCerulean and TRPV4-mVenus proteins were also characterized by western blots using both anti-TRPV4 antibody  and anti-GFP antibody  (data not shown). The mCerulean or mVenus fusion protein added to the recombinant TRPV4 did not alter the amino acid sequence at the C-ternimus of TRPV4, therefore leaving the epitopes intact for antibody recognition.Mouse TRPV4 coding sequence (Accession #NM_022017) was inserted between GSK1016790A (GSK101) was either a gift from GlaxoSmithKline or purchased from Sigma-Aldrich . GSK101 was dissolved in DMSO at 10 mM stock concentration and stored in aliquots at −20°C. It was then diluted into various working concentrations right before use.HeLa cells  were grown in RPMI medium supplemented with 10% FBS, 1 IU/ml penicillin and 1 µg/ml streptomycin in a 37°C and 5% CO2 incubator. HeLa cells were seeded onto glass coverslips or tissue culture dishes within 24 hrs of transfection, then an Effectene kit  was used to transfect HeLa cells with different plasmids. Cells were used for various experiments 20 to 24 hrs post transfection.2, 0.4 mM MgSO4, 3.3 mM NaHCO3, 2 mM CaCl2, 5.5 mM glucose, 10 mM HEPES, pH 7.4). The coverslip was attached to a perfusion chamber, which was then attached to the stage of an InCyt imaging workstation (Intracellular Imaging) for imaging of intracellular Ca2+ levels 2+-free experiments were carried out by washing away the Ca2+-containing MBSS with the Ca2+-free MBSS . Intracellular Ca2+ was calculated using the fura-2 fluorescence ratio method  which was converted to intracellular Ca2+ concentration as described by Gadzikowska et al. HeLa-TRPV4 cells grown on glass coverslips were loaded with 2 µM fura-2/AM for 1 hr at RT, then washed in the isotonic MBSS buffer /Delta GSK101] x 100%.To better compare changes in [Ca-t/τ, where Y is the current at time t, y0 is the residual current.Patch electrodes with a resistance of ≈2 MΩ were pulled from borosilicate micropipettes  using a micropipette puller  2+-containing extracellular solution consisted of 136 mMNaCl, 5.4 mMKCl, 0.5 mM MgCl2, 0.4 mM MgSO4, 3 mM NaHCO3, 2 mM CaCl2, 5 mM glucose and 10 mM HEPES . The Ca2+-free extracellular solution consisted of 145 mMNaCl, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose and 1 mM EGTA . The pipette internal solution contained 20 mMCsCl, 100 mM Cs aspartate, 1 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 1 mM Mg-ATP, and 0.1 mM Na-GTP . The holding potential was −90 mV for most experiments. The current-voltage relation was determined using a voltage ramp protocol from −100 to 100 mV over 200 msec.All experiments were performed at room temperature (≈25°C). Signals were filtered at 1 kHz, digitized at 10 kHz, and acquired using the Pulse program . The CaGSK101 stock in DMSO (10 mM) was diluted 1∶1000 in extracellular solution just before use and held in a series of independent syringes connected to an array of corresponding fused silica columns . The exchange of solutions was achieved by rapidly by shifting the tubes horizontally with a micromanipulator. The distance from the column mouth to the cell examined was about 100 µm. Cells in the recording chamber were continuously bathed in the extracellular solution. Each solution was delivered to the recording chamber by gravity.HeLa-TRPV4 cells were grown on 10-cm tissue culture plates until ready for harvest. The cells were rinsed twice with cold PBS, and scraped off of the plates using a spatula. Then a sucrose gradient-based membrane protein extraction kit  was utilized to purify plasma membrane proteins. Whole cell lysates were the intermediate products before the plasma membrane fraction was purified from the sucrose gradient. The final pellets containing pure plasma membrane proteins were achieved at the end of the extraction process. The resultant pellets were solubilized in a modified RIPA buffer  containing protease inhibitor cocktail . Protein concentrations were measured using the BCA method. Standard Western blotting  was performed to characterize and quantify TRPV4 channel expression. Primary antibodies used in this work include anti-TRPV4  for detecting TRPV4 as well as TRPV4-mVenus/Cerulean, anti-pan cadherin antibody  as internal loading control, and anti alpha-tubulin antibody  for verifying the purity of plasma membrane preparation. Densitometry  was utilized to quantify the abundance of a TRPV4 channel in the plasma membrane under different treatments. Normalized TRPV4 channel quantity was achieved by calculating the ratio of TRPV4/pan cadherin, as pan cadherin was the loading control for all the plasma membrane samples. All experiments were repeated 4 to 5 times.pre and mVenuspre, respectively). Acceptor photobleach was achieved by laser excitation at 514-nm set at 100% transmission for duration of 30 seconds (1 pulse/second). Then three post-bleach images of both mCerulean and mVenus were acquired . Regions of interest (ROI) were manually selected over the perinuclear region, designated as perinuclear (PN), or plasma membrane region, designated as membranous (M). A background ROI was also selected. The fluorescent intensity of the ROI was measured, background-subtracted and exported from the NIS software as an Excel file. The average of the pre-bleach and post-bleach fluorescence intensities were calculated from three images. The FRET efficiency was calculated as E =  (mCeruleanpost - mCeruleanpre)/mCeruleanpost. A chart graph was generated after averaging 50 to 80 ROI measurements from three sets of GSK101 time-course experiments.FRET microscopy was performed as described by Kenworthy group All data are presented as mean ± SE. Student t-test was used to compare the means between two treatment groups. P-values less than 0.05 were considered statistically significant.50 and Hillslope was chosen for calculation.A GSK101 dose-response curve was generated using the mean response (n = 4–6) at various GSK101 concentrations in SigmaPlot 10.0. The sigmoidal dose-response curve employing EC


However, the exact mechanisms underlying their effects or signaling pathways mediated by FTY720 have not been completely established. To gain further insights into molecular mechanisms of FTY720 action, the effect of FTY720 on Ca2+ signaling in fission yeast was analyzed. The addition of Ca2+ enhanced the sensitivity induced by FTY720, and mutants lacking genes required for calcium homeostasis, including calcineurin and its downstream transcription factor, Ppb1-responsive zinc finger protein (Prz1), were hypersensitive to FTY720 and CaCl2. The effect of FTY720 on calcineurin signaling was monitored by utilizing a luciferase reporter construct fused to three tandem repeats of the calcineurin-dependent response element (CDRE), which gives an accurate measure of calcineurin activity. The addition of FTY720 increased calcineurin activity as well as Ca2+ influx in a concentration-dependent manner. Notably, the FTY720-mediated Ca2+ influx and calcineurin activation were reduced markedly by the deletion of yam8+ or cch1+ encoding putative subunits of a Ca2+ channel. Consistently, the deletion of Pmk1 mitogen-activated protein kinase (MAPK), which plays an important role in the activation of the Yam8/Cch1 channel, markedly decreased the intracellular Ca2+ levels upon FTY720 treatment. These results suggest that the FTY720-stimulated Ca2+/calcineurin signaling activation partly involves the Yam8/Cch1 channel in fission yeast.Fingolimod hydrochloride (FTY720) is the first in class of sphingosine 1-phosphate (S1P) receptor modulator approved to treat multiple sclerosis via down-regulation of G protein-coupled S1P receptor 1 by its phosphorylated form (FTY720-P). Many studies have revealed that FTY720 exerts various biological effects, including antitumor activities, angiogenesis inhibition, Ca Isaria sinclairii [Fingolimod hydrochloride FTY720) is a novel sphingosine 1-phophate (S1P) receptor modulator that was found by chemical modification of myriocin, a natural product isolated from culture filtrates of the ascomycete 0 is a nonclairii . AlthougInterestingly, in addition to its therapeutic use as an immunomodulating drug, FTY720 was also shown to exert potent antitumor and antimetastatic activities in different tumor types, including breast cancer, bladder cancer, hepatocellular carcinoma, and leukemia ,5. SeverAccumulating evidence also suggests that FTY720 may exert some of these effects independently of S1P receptors by modulating a range of other recently described proteins targeted by nonphosphorylated FTY720 . For exaSchizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae are among the simplest eukaryotic organisms that are widely used as valuable tools for the study of basic cellular processes and pathways relevant to higher eukaryotes, including mechanisms of cell cycle control, metabolism, and membrane trafficking [This study uses fission yeast as a model eukaryotic system to dissect the biological activity of FTY720. The fission yeast fficking ,14. Bothfficking -18. In bfficking  and that2+/calcineurin signaling was analyzed. In fission yeast, a mutation in ppb1+ encoding the Ca2+/calmodulin-dependent protein phosphatase [prz1+ encoding a transcription factor downstream of calcineurin involved in Ca2+ homeostasis [2+ homeostasis results in increased sensitivity to FTY720. We also demonstrated that FTY720 activates calcineurin signaling by stimulating Ca2+ influx mediated by the Yam8/Cch1 Ca2+ channel in fission yeast. To better understand the signaling pathways mediated by FTY720, the effect of FTY720 on Casphatase ,21 as weeostasis  affectedpombe strains used in this study are listed in ppb1::ura4+). In addition, gene knockouts are abbreviated by the gene, which is preceded by Δ . Proteins are denoted by Roman letters, and only the first letter is capitalized . Genome DNA clones were provided by the National Bio Resource Project, Yeast Genetic Resource Center . The S. FTY720 and FTY720-P (phosphorylated FTY720) were kindly received as gifts from the Mitsubishi Tanabe Pharma Corporation . Yeast growth media containing each of these chemicals were prepared by mixing stock solutions of FTY720 or FTY720-P with the YPD or YES medium to achieve the desired drug concentration. For agar media, the stock solution of the appropriate drug was added after autoclaving and cooling of the media to approximately 50°C. For FTY720, the stock solution was prepared using water, whereas for FTY720-P, the stock solution was prepared using 80% ethanol containing 10 mM NaOH . When coLight microscopy methods  used to observe the localization of GFP-Prz1 was performed as described previously . All the image quantifications were performed for three individual datasets, which summed up to 300 counted cells. The mean and the standard deviation (SD) of the relative light units (RLU) values for three different experiments were calculated for each sample. Data were analyzed using a two-way ANOVA, followed by a posthoc test using Williams’ multiple comparison.AGCCTC or GAGGCT) in pKB5723 with the mutant CDRE oligonucleotide  that contains three tandem repeats of the mutated CDRE , thereby yielding the multicopy 3×mtCDRE::luc(R2.2) reporter vector pKD2767. Calcineurin-dependent response element (CDRE)-reporter activity in living cells was monitored by the multicopy 3×CDRE::luc (R2.2) reporter vector pKB5723 as described previously . We also2+ levels were determined using a previously described method with minor modifications [3 LB960: Berthold) at 30-s intervals.Cytoplasmic Caications . In brie2+ signaling and FTY720 sensitivity, the effect of Ca2+ on the growth of wt cells in the medium supplemented with FTY720 was first tested. As shown in 2 alone only marginally affected the growth of wt cells in the solid YPD medium. However, the same concentration of CaCl2 exacerbated the growth of wt cells treated with various concentrations of FTY720 . In fission yeast, calcineurin dephosphorylates and activates Prz1, a zinc finger-type transcription factor involved in Ca2+ homeostasis [prz1 cells failed to grow in media containing more than 20 μM FTY720 (prz1). Notably, these mutant cells also displayed enhanced sensitivities to CaCl2 compared with the wt cells because they failed to grow in the medium containing more than 80 mM CaCl2 . The addition of 200 mM CaCl2 exerted similar effects on GFP-Prz1 localization . Thus, FTY720 stimulates nuclear translocation of Prz1 as efficiently as Ca2+.The above findings prompted the examination of the effect of FTY720 on calcineurin/Prz1 signaling. The intracellular localization of GFP-Prz1 was examined because the activation of calcineurin causes the translocation of GFP-Prz1 from the cytoplasm to the nucleus . The add2 (50–200 mM). FTY720 treatment caused a dose-dependent increase in the 3×CDRE::luc (R2.2) response, exhibiting a peak rise and then approaching a constant level (ppb1) and prz1-null (Δprz1) cells treated with 10 μM FTY720, the reporter response both in the absence  and the presence of FTY720 was reduced markedly ), which was established as an accurate measure of the calcineurin activation in living cells . The wt markedly , consistAGCCTC in the CDRE was mutated to AGATCT . The mutation in CDRE dramatically decreased the reporter activity upon FTY720 and CaCl2 treatment (2 was exerted via Prz1 binding to the CDRE motif.The mutant version of the 3×CDRE reporter plasmid 3×mt CDRE::luc (R2.2) was next created, in which the consensus motif While this range of concentration is higher than that required for immune modulation 10-100 nM) in cultured mammalian cells, it should be noted that significantly higher concentrations of FTY720 were used (5-25 μM) when examining the inhibition of tumor development, induction of apoptosis and stimulation of the intracellular concentration of calcium ions in mammalian cells ,10,29-32-100 nM i2+ influx reportedly induce calcineurin activation [2+ levels upon FTY720 treatment were monitored in fission yeast, using adh1-GFP-19-AEQ, which have been established as a sensitive method to monitor the cytoplasmic Ca2+ levels [2+ levels, which rapidly increased and reached a peak level immediately after the addition of FTY720, rapidly decreased thereafter and exhibited a less pronounced second peak, and then reached a steady state level  was examined in the same assay. The results demonstrated that the peak responses and increase in cytoplasmic Ca2+ levels elicited by FTY720 were almost inhibited by the addition of EGTA to the culture medium . The addition of CaCl2 induced the additive response of calcineurin activity upon FTY720 treatment  chloride (GdCl3), on Ca2+ influx induced by FTY720 was examined. GdCl3 reduced the peak response of Ca2+ levels upon FTY720 treatment in a dose-dependent manner  also stimulates Cat manner , FTY720.t manner . In addit manner . Finallyt manner . In addit manner . pombe cell wall as well as the possibility that the compound might be more rapidly metabolized in S. pombe cells than in animal cells. Because FTY720 was reported to be transported across cellular membranes with ATP-binding cassette (ABC) transporter family members [2+ signaling could be due to rapid export of the compound from the cells. We then tested the intracellular Ca2+ levels induced by FTY720-P in cells lacking both bfr1+ and pmd1+, encoding two major S. pombe drug-efflux transporters, which were involved in multidrug resistance [2+ levels in wild-type cells  and that the difference in intracellular Ca2+ levels upon FTY720 stimuli (50 μM) between wt and Δbfr1Δpmd1 null cells were significant, with a P-value of < 0.01 (##) , as previously reported in Saccharomyces cerevisiae [Fission yeast cells also have the homolog of sphingoid long-chain base (LCB) kinase, pSPHK1 (accession no. T38776) that catalyzes the phosphorylation of LCBs to form LCB 1-phosphate in mammals . Howeverrevisiae ,20, whic2+ channel protein Yam8/Cch1 underscores the existence of a specific mechanism for FTY720-dependent calcium signaling in fission yeast. Whether FTY720 can traverse the yeast cell wall and plasma membrane to act intracellularly, or affects cell integrity, thereby activating Yam8/Cch1 channel remains unknown. However, we favor the former possibility based on the lipophilic nature of FTY720, as well as our findings that an anti-fungal agent such as miconazole, or detergent such as 1% Tween 20, or an ER-stress-inducing compound DTT, failed to induce Ca2+ influx as efficiently as FTY720 did (our unpublished results). In addition, in the presence of extracellular Ca2+-chelators such as EGTA, FTY720 was unable to stimulate Ca2+ influx. Therefore, no evidence for FTY720-stimulated Ca2+ release from the internal store has been obtained in fission yeast thus far. It should be noted however, that EGTA addition failed to rescue the growth inhibition of wild-type cells by FTY720 (2 (up to 100 mM) failed to rescue the growth inhibition of Δyam8, Δcch1, Δyam8Δcch1 cells, thus suggesting that FTY720-induced cell growth defect may not be explained only by activation of Ca2+/calcineurin signaling, and may instead represent a broader effect. We therefore hypothesized that although the disturbance of appropriate regulation of the Ca2+/calcineurin signaling resulted in increased sensitivity of fission yeast proliferation to adapt to physiological responses induced by FTY720, and that Yam8/Cch1 plays an important role on calcineurin activation induced by FTY720, it may not support a direct connection between calcineurin activity and inhibition of proliferation. Because fission yeast cells also have the homolog of sphingoid long-chain base (LCB) kinase, pSPHK1 (accession no. T38776) that catalyzes the phosphorylation of LCBs to form LCB 1-phosphate in mammals [2+/calcineurin signaling in fission yeast.The involvement of the Cay FTY720 . In addi mammals , it woul2+/calcineurin signaling pathway plays an important role in various cellular functions, including immune regulation, cardiac hypertrophy and apoptosis, and FK506 and CsA are specific inhibitors of calcineurin both in humans and yeasts. Notably, the findings presented in this study suggested the cross-talk between FTY720-mediated and FK506-regulated signaling pathway, both of which are involved in immune regulation. In clinical organ transplantations, FK506-based combination therapy with other immunosuppressants is widely used to reduce the side effects of individual drugs. Notably, the combination therapy of FTY720 with CsA or FK506 has a marked prolonging effect on allograft survival as compared with the monotherapy of FTY720, CsA, or FK506 [2+ signaling pathway. The CaFigure S12+Effect of vehicle on the basal intracellular Calevels. Left panel: The wild-type cells harboring adh1-GFP-19-AEQ were treated with water and ethanol containing NaOH, and experiments were performed as described in (TIF)Click here for additional data file.Figure S2Effect of FTY720-P on the intracellular localization of GFP-Prz1. Translocation of GFP-Prz1 to the nucleus is induced by FTY720 addition, but not by FTY720-P. Wild-type cells expressing GFP-Prz1 were grown in EMM medium at 27°C and analyzed by fluorescence microscopy as described in (TIF)Click here for additional data file.Figure S32+ or EGTA on inhibition of proliferation by FTY720 in various strains.The effect of Ca A serial dilution assay of the wild-type (wt), Δyam8, Δcch1, and Δyam8Δcch1 cells grown in rich YPD medium containing the indicated concentrations of FTY720, CaCl2 and EGTA.(TIF)Click here for additional data file.


Their neurotoxicity involves dysregulation of calcium ion (CaWe aimed to define the structure–activity relationship (SAR) for PBDEs and their metabolites toward ryanodine receptors type 1 (RyR1) and type 2 (RyR2) and to determine whether it predicts neurotoxicity.3H]ryanodine binding, microsomal Ca2+ fluxes, cellular measurements of Ca2+ homeostasis, and neurotoxicity to define mechanisms and specificity of PBDE-mediated Ca2+ dysregulation.We analyzed [ortho-bromine substituents and lacking at least one para-bromine substituent  activate RyR1 and RyR2 with greater efficacy than corresponding congeners with two para-bromine substitutions . Addition of a methoxy group in the free para position reduces the activity of parent PBDEs. The hydroxylated BDEs 6-OH-BDE-47 and 4′-OH-BDE-49 are biphasic RyR modulators. Pretreatment of HEK293 cells (derived from human embryonic kidney cells) expressing either RyR1 or RyR2 with BDE-49 (250 nM) sensitized Ca2+ flux triggered by RyR agonists, whereas BDE-47 (250 nM) had negligible activity. The divergent activity of BDE-49, BDE-47, and 6-OH-BDE-47 toward RyRs predicted neurotoxicity in cultures of cortical neurons.PBDEs possessing two ortho and para position determined whether a congener enhanced, inhibited, or exerted nonmonotonic actions toward RyRs. These results identify a convergent molecular target of PBDEs previously identified for noncoplanar polychlorinated biphenyls (PCBs) that predicts their cellular neurotoxicity and therefore could be a useful tool in risk assessment of PBDEs and related compounds.We found that PBDEs are potent modulators of RyR1 and RyR2. A stringent SAR at the  Worldwide use of chemically stable polybrominated diphenyl ethers (PBDEs) as flame retardants in consumer products has increased since the 1970s . Levels ortho position, in particular, noncoplanarity of their phenyl rings. Like the ortho-substituted PCBs (2+) signaling in several cell types   membranes where they contribute to and regulate essential aspects of Ca2+ signaling  that have more than one chloro-substituent in the ted PCBs , PBDEs  . For exaarations , and altarations . Developarations . In ratsarations . Identifignaling .Para- and ortho-substitutions are a key structural determinant of this activity and are predictive of their neurotoxicity.In the present study we examined the structure–activity relationship (SAR) of PBDEs and their metabolites toward the two major RyR isoforms found in the brain and striated muscle (RyR1 and RyR2). Our findings indicate that PBDEs are potent modulators of both RyR isoforms. 3H]Ryanodine  was obtained from Perkin-Elmer New England Nuclear . 4-Chloro-m-cresol (4-CmC), diphenyl ether (DE), rapamycin, and Ca2+ ionophore A23187 were purchased from Sigma-Aldrich .We purchased neat certified 2,2′-dibromodiphenyl ether (BDE-4), 4,4′-dibromodiphenyl ether (BDE-15), 2,2′,4-tribromodiphenyl ether , 2,2′,3,4′-tetrabromodiphenyl ether , BDE-47 (100% pure), 2,2′,4,5′-tetrabromodiphenyl ether , the hydroxylated BDEs 6-OH-BDE-47 (98.6% pure) and 4′-OH-BDE-49 (97.8% pure), and PCB-95 (99.1 ± 4% pure) from AccuStandard  and verified purity and composition by gas chromatography-mass spectrometry through the Superfund Research Program Analytical Core. Methoxy-derivatives were provided by Å. Bergman . Ry binding to microsomal membranes in the absence and presence of PBDE or their metabolites, a measure of how RyR activity is affected, as previously described  were determined by curve fitting of three experiments.We measured PBDE-induced release of Caescribed . See Supnull] cells in culture and the protocols for transfection and selection protocols for HEK293 cells that stably express RyR1 (HEK293RyR1) or RyR2 (HEK293RyR2) as described previously . See Supplemental Material (doi:10.1289/ehp.1002728) for additional details.We followed the method for growing human embryonic kidney  assay to test cell viability in cultured mouse cortical neurons after 48-hr exposures to BDE-47, BDE-49 or 6-OH-BDE-47 using the CellTiter 96 Cell Proliferation Assay kit  as previously described . See Sup3H]Ry binding analysis to microsomes. Neither unsubstituted diphenyl ether nor BDE-15 (< 20 μM) altered the amount of specific binding of [3H]Ry to RyR1, a measure of receptor activation  by bolus additions of Ca2+ to the assay buffer containing ATP (Ca2+-loading phase). After the loading phase was complete , addition of BDE-4 (1–20 μM) elicited a net release of Ca2+ . BDE-4–triggered Ca2+ release was fully blocked by ruthenium red , an RyR blocker .Antipyrylazo-III is a low-affinity membrane impermeable dye that allows quantification of Cavesicles . Spectro blocker . EC50 vantration . BDE-15 2+ channel by ortho-substituted PCBs requires an intact association of RyR1 with its accessory protein FK506-binding protein 12 kDa (FKBP12) (3H]Ry binding to RyR1 by BDE-4 (5 μM) was inhibited by inclusion of rapamycin in the assay (2 min preincubation) to disrupt the RyR1-FKBP12 complex  to vesicles actively loaded with Ca2+ caused release of accumulated Ca2+, and this response was inhibited in vesicles pretreated with rapamycin in a concentration-dependent manner (2+ release without inhibiting caffeine-induced Ca2+ release (Activation of the Ca(FKBP12) . Consist complex . The impt manner . Rapamyc release , indicatpara-chloro-substitutions of PCBs significantly influence activity toward RyR1 , whereas BDE-49 and BDE-17 enhanced binding > 450% relative to vehicle control (EC50 = 2 μM). BDE-42 showed intermediate activity (250% of vehicle control at 10 μM) , and BDE-47 had negligible activity. A similar SAR was identified with [3H]Ry binding to cortical membrane preparations that contain a mixture of RyR1 and RyR2 . Collectively, these data indicate that in addition to two ortho-bromines, one unsubstituted para position is critical for enhancing RyR activity.Because ard RyR1 , we invet 10 μM) . A similt 10 μM) . At concpara substitution is an important determinant of RyR activity, we examined the activity of para-methoxy metabolites using [3H]Ry binding and Ca2+ flux analyses of RyR1. Addition of a methoxy substituent at the 4 or 4′ positions of BDE-17, BDE-42, or BDE-49 significantly reduced the apparent efficacy by > 2-fold in [3H]Ry binding experiments . However, addition of methoxy to the 5 position of BDE-47 did not alter activity toward RyR1. Addition of BDE-49 (10 μM) subsequent to loading vesicles with Ca2+ in the presence of ATP elicited a very robust efflux of the accumulated Ca2+, and the release could be completely blocked by the RyR channel blocker RR . However, regardless of the pretreatment protocol, the RyR1 channels remained responsive to 4-CmC .As predicted by results with BDE-4 , microso3H]Ry binding analysis , and this effect was completely blocked by RR  and HEK293 cells, which stably express either RyR1 (HEK293RyR1) or RyR2 (HEK293RyR2) . Cells of each genotype were pretreated with 250 nM BDE-49 or BDE-47 for 16 hr before loading them with the Ca2+-sensitive dye Fluo-4. Once loaded with Ca2+ indicator, cells were imaged to detect changes in cytoplasmic Ca2+ ([Ca2+]i) before and after challenge with RyR agonists caffeine or 4-CmC (RyR1 responded to brief (10-sec) focal application of caffeine with a Ca2+ transient whose amplitude was concentration dependent  at lower caffeine concentrations  in contrast to HEK293null cells . HEK293RyR2 cells pretreated for 16 hr with BDE-49, but not BDE-47, showed significantly larger Ca2+ transient amplitudes  compared with vehicle controls .To determine whether the different efficacies of BDE-47 and BDE-49 toward RyRs extend to RyR-dependent signaling events in intact cells, we tested the activity of the two congeners toward HEK293or 4-CmC . HEK293Rin vitro . MEAs have been used increasingly to assess altered development of neural networks and excitotoxicity caused by xenobiotics . Corticain vitro . Sequentin vitro .p < 0.05). A potentially significant finding is that 6-OH-BDE-47 (10 μM), a major metabolite of BDE-47, also caused significant loss of cell viability (We next determined whether exposures to BDE-47 and BDE-49 at higher concentrations (low micromolar) and for longer times (48 hr) influence the viability of cortical neuron cultures. Compared with vehicle controls, cultures exposed to 5 and 10 μM BDE-49 invariably showed loss of phase-bright cell bodies, decreased soma diameter, extreme fasciculation of processes, and a tendency for the monolayer to pull up off the substrate . In contiability .in utero or postnatal exposure to PBDEs resulted in hyperactivity in rats , each of which has been shown to alter Ca2+ homeostasis, in part by dysregulating RyR channels (3Rs), are a family of Ca2+ channels in endoplasmic or sarcoplasmic reticulum, broadly expressed in both excitable cells  and nonexcitable cells   . RyRs ashocytes) . Environhocytes) .ortho- and para-bromine substituents significantly influence the level of activation of RyRs, as highlighted by divergent activities of BDE-47 and BDE-49, and is unrelated to differences in lipophilicity. In this regard, the free concentration of BDE-49 is likely to be significantly lower than those added to the assays used in this study. This aspect of SAR is similar to that observed with the influence of para-chlorines on PCB activity toward RyR1 and RyR2  and apparently fails to detect the initial channel activation elicited by 6-OH-BDE-47. However Ca2+ flux measurements clearly show that the immediate actions of this congener are activation of RyR1 and release of Ca2+ accumulated in microsomal vesicles, whereas prolonged exposure significantly attenuates 4-CmC–induced Ca2+ release. Our results reveal that OH-PBDEs have complex biphasic actions on RyR function and SR/ER Ca2+ transport properties that depend not only on the local concentration but also on the length of the exposure.The results from the present study showing that 6-OH-BDE-47 produces temporally defined biphasic actions on microsomal CaIanthella basta, termed “bastadins”  PC12 cells. A high concentration of BDE-47 (20 μM) enhanced the Ca2+ transient amplitude triggered by bolus addition of extracellular K+, whereas other PBDE congeners lacked significant influence (2+ transient amplitude. These previously reported effects of PBDEs and OH-PBDEs could be explained, at least in part, by their actions on RyR channels as we describe here. Sensitization of RyRs in resting cells by OH-PBDEs could result in chronic Ca2+ leakage and depletion of ER stores. This could account in large part for both the rapid and delayed rises in cytoplasmic Ca2+ previously observed by 2+ transient amplitude triggered by depolarization subsequent to 20 min pretreatment with 2 and 20 μM OH-PBDE (3H]Ry binding studies, potently sensitizes caffeine- or 4-CmC–triggered Ca2+ release in HEK293 cells that express RyRs, and is a potent excitotoxicant toward primary cortical neurons. In this regard, 48 hr exposure to BDE-47 < 10 μM does not promote loss of neuronal viability, whereas BDE-49 does. These observations are apparently at odds with reports that developmental exposure to BDE-47 causes developmental neurotoxicity in rodent models (in vivo are mediated by 6-OH-BDE-47 rather than the parent congener; the lack of effect of the parent compound on either spontaneous activity or cell viability in cultured cortical neurons reflects the limited metabolic capacity of this in vitro model.Our results with OH-BDEs suggest an underlying mechanism for the results reported by nfluence . Interes OH-PBDE  would bet models . One int3H]Ry binding studies are a rapid means of defining SARs that predict neurotoxic PBDEs, much like its use to identify neurotoxic PCBs (The present study indicates that [xic PCBs . The toxxic PCBs  that BDExic PCBs , includixic PCBs . The mec2+ signaling dysregulation mediated by RyR channel mechanisms should be included in assessments of risk.The present study is the first to identify RyR1 and RyR2 as direct targets of both PBDEs and their hydroxylated metabolites. These results are significant because RyRs are broadly expressed in excitable and nonexcitable cells, where they regulate key physiological and pathophysiological functions . Certain