In the present study we investigated whether elevating [Ca2+]o induced store-operated calcium entry (SOCE) in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca2+]o to osteoblastic proliferation.The local concentration of extracellular Ca2+ concentration ([Ca2+]c) of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR) as well as inhibitors of phospholipase C (PLC), SOCE and voltage-gated calcium (Cav) channels were applied to study the mechanism in detail.Cytosolic Ca2+]o evoked a sustained increase of [Ca2+]c in a dose-dependent manner. This [Ca2+]c increase was blocked by TMB-8 (Ca2+ release inhibitor), 2-APB and BTP-2 (both SOCE blockers), respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist) or U73122 (a PLC inhibitor) strongly reduced the [Ca2+]o-induced [Ca2+]c increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca2+]o resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca2+]o significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM , 2-APB, BTP-2, TMB-8, NPS2143 and U73122, respectively, but not affected by Cav channels antagonists.Our data showed that elevating [Ca2+]o induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca2+ concentration.Elevating [Ca Reducing formation or increasing resorption may lead to bone loss, osteoporosis, eventually debilitating fractures 2+]c) increase which is critical in regulating a variety of cellular functions including secretion, apoptosis, and more specifically proliferation 2+ concentration in the intracellular endoplasmic reticulum (ER) stores. Under physiological conditions, receptor-mediated activation of the phospholipase C (PLC) induces the generation of inositol 1,4,5-trisphosphate (IP3) and subsequently triggers IP3 receptor-related Ca2+ release from ER, which may stimulate SOCE in turn 2+]o-induced osteoblastic proliferation, the underlying intracellular signaling is largely unclear. Especially, it remains unknown whether the elevation of [Ca2+]o can induce SOCE, and whether high [Ca2+]o-induced osteoblastic proliferation is conducted through SOCE in osteoblasts.In various cell types, the store operated calcium entry (SOCE) determines sustained cytosolic calcium concentration , a member of G-protein coupled receptor family 2+ release through PLC/IP3 pathway 2+ release could trigger SOCE in breast cancer cells and cardiomyocytes 2+ influx in renal collecting duct cells 2+]o obviously induced a sustained rise of [Ca2+]c in rat calvarial osteoblasts. Therefore, the aim of this study was to investigate the mechanism of [Ca2+]c increase induced by [Ca2+]o in rat calvarial osteoblasts. We asked whether the effects of [Ca2+]o on [Ca2+]c depended on the activation of CaSR-related PLC/IP3 signaling and SOCE. Furthermore, we examined the contribution of [Ca2+]c increase to high [Ca2+]o-induced proliferation in primary rat calvarial osteoblasts.It was established that extracellular Cathe Guide, NRC 2011), and it was also approved by the Institutional Animal Care and Use Committee at Nankai University .The animal protocol in this study conformed to the Guide for the Care and Use of Laboratory Animals (New born Wistar rats (3-day-old) were obtained from Academy of Military Medical Sciences . DMEM and fetal bovine serum (FBS) were from Gibco (USA) and HyClone (USA), respectively. Fura-2/AM was purchased from Biotium (USA). The rest of reagents, including trypsin, collagenase II, EGTA, DMSO, thapsigargin (TG), BAPTA-AM, TMB-8, 2-APB, BTP-2 (YM-58483), U73122, U73343, NPS2143, spermine, nifedipine and verapamil were purchased from Sigma-Aldrich (USA). CellTiter 96 AQueous One Solution Cell Proliferation Assay kit and CellTiter-Glo Luminescent Cell Viability Assay kit were purchased from Promega (USA).2.Rat calvarial osteoblasts were isolated and cultured as previously described 2 2 mM, MgCl2 1 mM, glucose 10 mM and HEPES 10 mM, pH  = 7.4) for 1 h at room temperature. After washing extensively with HBSS, cells were bathed in fresh HBSS solution. [Ca2+]c was measured with calcium imaging system built on an inverted fluorescence microscope (Olympus IX51). The Ca2+ indicator fura-2 was alternately excited at 340 nm and 380 nm with a Lambda 10–2 sutter. Fluorescence images (filtered at 515 nm±25 nm) were captured by a CCD camera (CoolSNAP fx-M) and quantitated with MetaFluor. [Ca2+]c was represented by the ratio of fluorescence intensity at 340 nm/fluorescence intensity at 380 nm (F340/F380). At least three independent experiments were done for each condition. One curve of calcium changes was plotted as the representation of other similar traces. Ca2+-free HBSS solution was made by substituting MgCl2 for CaCl2 at the same concentration.Osteoblasts were loaded with 5 µM fura-2/AM in Hanks’ balanced salt solution (HBSS)  with DMEM containing 5% FBS at 37°C. Then, the pretreated-cells in each dish were monitored by an inverted light microscope (Olympus IX51) at 0, 24, 48 and 72 h in turn. In the meantime, the cell numbers in each dish were measured from at least five regions (1 mm×1 mm grids) at the indicated time. For MTS and ATP assays, osteoblasts were seeded into 96-well plate at ∼1×104 cells/well at 37°C in DMEM with 5% FBS and incubated overnight before treating with or without test agents for 72 h. The MTS assay was performed by directly adding 20 µl of the AQueous One Solution Reagent to culture wells (100 µl/well), incubating for 4 h and then recording the absorbance at 490 nm (A490) with an ELISA reader (Bio-Rad Imark Microplate Reader). The ATP assay was carried out by adding 100 µl of the CellTiter-Glo Reagent (Buffer plus Substrate) to each well, then mixing contents for 2 minutes on an orbital shaker to induce cell lysis. After that the plate was incubated for 10 minutes to stabilize luminescent signal. The luminescent signal was measured by a luminometer . The ATP concentration in each well was derived from the standard curve.The proliferation of osteoblasts was assessed by morphological observations and direct cell counting. The number of viable cells in proliferation was further determined by MTS assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay kit) and ATP assay (CellTiter-Glo Luminescent Cell Viability Assay kit), respectively. For morphological observations, osteoblasts were plated in 35 mm culture dishes  were calculated according to the dose-response curve fitting with the logistic equation: Y is the response, maxY is the asymptotic maximum, minY is the asymptotic minimum, x is the extracellular calcium concentration and n is the Hill coefficient.All data passed the normality test and were presented as mean ± standard deviation. The statistical comparison between two groups was carried out using Student’s t-test (Origin 8.0), and the analysis for multiple groups was using Dunnett’s test . 2+-pump blocker thapsigargin (TG), a drug widely used to test SOCE. It was seen from 2+]c rise mediated by Ca2+ release from Ca2+ stores with nominally Ca2+-free HBSS. Adding 2 mM CaCl2 after [Ca2+]c returning to the basal level triggered [Ca2+]c increase due to Ca2+ entry. This Ca2+ entry was strongly inhibited by the application of potent SOCE blockers 2-APB (25 µM) vs. 0.30±0.02 after application of 2-APB for 100 s, P<0.05) or BTP-2  vs. 0.38±0.10 after application of BTP-2 for 100 s, P<0.05) during the high [Ca2+]c plateau evoked by adding 2 mM CaCl2 (2+ entry was evidently abolished when cells were pretreatment with 2-APB (25 µM) or BTP-2 (20 µM) before adding TG  2+]c increase evoked by elevating [Ca2+]o (10 mM) as show in 2+]c increase were not different from that of control  channels 2+]o-induced [Ca2+]c increase, a series of experiments were carried out. Firstly, the rise of [Ca2+]c induced by 10 mM [Ca2+]o was decreased significantly  or 20 µM BTP-2  (2+]o induced the activation of SOCE underlying the increase of [Ca2+]c.To investigate whether SOCE was associate with [Ca P<0.05) . Taken t3 signaling in [Ca2+]o-induced SOCE was examined in the following experiments. Firstly, the elevating [Ca2+]o-induced [Ca2+]c increase was almost abolished when cells were pretreated with a specific CaSR antagonist NPS2143 (10 µM) vs. 0.024±0.004 for NPS2143, P<0.05; 2+]c rise significantly  and BTP-2 (20 µM) suppressed the sustained [Ca2+]c increase induced by spermine in Ca2+-containing HBSS (2+]c increase by spermine in the presence of NPS2143 (10 µM) or U73122 (5 µM)   in Ca2+-2+]o on the proliferation capacity of osteoblasts and the contribution of [Ca2+]o-induced [Ca2+]c increase to proliferation of osteoblasts, we carried out a series of experiments to assess the cell proliferation in different levels of [Ca2+]o with or without various inhibitors by morphological observations and cell counting, as well as estimating the proliferating activity by MTS and ATP assays. Morphological images of osteoblasts were obtained with medium containing 1.8, 3, 5, 10 mM [Ca2+]o at 0, 24, 48 and 72 h, respectively. It was found that the cell numbers in medium with higher [Ca2+]o were increased significantly in comparison with control  in a concentration-dependent and time-dependent manner (490) (MTS assay)  (2+]o-stimulated cell proliferation was decreased significantly in the presence of 2-APB (25 µM), BTP-2 (20 µM), TMB-8 (50 µM), NPS2143 (10 µM) and U73122 (5 µM), respectively, while treating osteoblasts with U73343 (5 µM), nifedipine (10 µM) or verapamil (10 µM) had little influence on cell proliferation   and ATP S assay)  were incM (2 µM) . Furtherferation  and S2. 2+]o triggered [Ca2+]c increases in a dose-dependent manner with a EC50 of 5.4±1.2 mM in rat calvarial osteoblasts. This [Ca2+]c increase was abolished by SOCE blockers 2-APB and BTP-2, or Ca2+ release inhibitor TMB-8, while not affected by Cav channels antagonists nifedipine or verapamil. Furthermore, specific CaSR antagonist NPS2143 or PLC inhibitor U73122 strongly reduced the [Ca2+]c increase resulted from elevating [Ca2+]o. These data indicated that elevating [Ca2+]o induced SOCE in osteoblasts, which was dependent on the activation of CaSR and PLC. The SOCE route, rather than Cav channels contributed to elevation [Ca2+]o-induced [Ca2+]c increase. In addition, high levels of [Ca2+]o promoted the proliferation of osteoblasts. This promotion was also significantly inhibited by 2-APB, BTP-2, TMB-8, NPS2143 and U73122, respectively, suggesting a contribution of CaSR-related PLC/IP3-SOCE pathway to high [Ca2+]o-induced proliferation in osteoblasts.In the present study, we found that elevating o triggered a sustained [Ca2+]c increase  channels especially TRPC subfamily were considered for candidate SOCE machinery in osteoblasts as well as other cell types. Many members of TRP channels had been identified to exist in osteoblasts and served as SOCE machinery 2+]o-triggered [Ca2+]i increases needs further investigation.The local concentration of extracellular Caincrease . This o-induced [Ca2+]c increase was strongly blocked by specific CaSR antagonist NPS2143 and PLC inhibitor U73122. Similar responses were observed when the cells were stimulated with spermine, a specific CaSR agonist  and ERK2, but other mitogen-activated protein (MAP) kinase signal pathways, p38 MAP kinase and SAPK/JNK, were not activated by [Ca2+]o in osteoblasts 2+]o in human dental pulp cells 2+]o was required for phosphate-dependent ERK1/2 phosphorylation and regulation of mineralization-associated genes in osteoblasts 2+]c increase in various cell types including embryonic stem cells and smooth muscle cells 2+]o-induced proliferation of osteoblasts. In this study, our data showed that intracellular calcium chelator BAPTA-AM reversed the high [Ca2+]o-induced proliferation. 2-APB, BTP-2, TMB-8, NPS2143 or U73122 also reduced the [Ca2+]o-induced proliferation  Statistic data of ATP concentration in each group. The quantitation of the ATP concentration assessed by ATP assay is proportional to the number of viable cells present in culture. Osteoblasts were incubated for 72 h in culturing medium with different levels of [Ca2+]o or in a medium with 2 µM BAPTA-AM+10 mM [Ca2+]o (n = 7 for each case), * showed P<0.05, compared with [Ca2+]o  = 1.8 mM group; # showed P<0.05, compared with [Ca2+]o  = 10 mM group. (B) Statistic data of ATP concentration measured after culturing for 72 h in [Ca2+]o = 10 mM medium alone or together with 2-APB (25 µM), BTP-2 (20 µM), TMB-8 (50 µM), NPS2143 (10 µM), U73122 (5 µM), U73343 (5 µM), nifedipine (10 µM) and verapamil (10 µM) (n = 5 for each case), respectively. * showed P<0.05 in comparison with [Ca2+]o  = 10 mM group.(TIF)Click here for additional data file.Figure S22+]o-induced increase in cell numbers was blocked by inhibitors including 2-APB, BTP-2, TMB-8, NPS2143 and U73122, respectively, but not affected by voltage-gated calcium channels blockers nifedipine and verapamil in rat calvarial osteoblasts.High [Ca Osteoblasts were cultured in medium with 10 mM [Ca2+]o alone or together with 2-APB (25 µM), BTP-2 (20 µM), TMB-8 (50 µM), NPS2143 (10 µM), U73122 (5 µM), U73343 (5 µM), nifedipine (10 µM) and verapamil (10 µM). Representative cell morphological images were captured at 0 h, 24 h, 48 h and 72 h using a 10× objective.(TIF)Click here for additional data file.


Calcium ions are inter- and intra-cellular messengers that are involved in numerous signalling pathways. Fluctuating compartment-specific calcium ion concentrations can lead to localised and even plant-wide oscillations that can regulate downstream events. Understanding the mechanisms that give rise to these complex patterns that vary both in space and time can be challenging, even in cases for which individual components have been identified. Taking a systems biology approach, mathematical and computational techniques can be employed to produce models that recapitulate experimental observations and capture our current understanding of the system. Useful models make novel predictions that can be investigated and falsified experimentally. This review brings together recent work on the modelling of calcium signalling in plants, from the scale of ion channels through to plant-wide responses to external stimuli. Some  Numerous stimuli lead to changes in calcium concentrations that regulate plant responses, so obtaining insights into how plants adapt to their environment often requires understanding the processes that govern calcium levels within and between compartments. Many processes are, however, so complex that our understanding is hindered by a breakdown of intuition. Such complexity can arise already with three or more variables for non-linear interactions, as are typical for biological systems. Building simplified mathematical models that capture the key characteristics of the system under study is in such cases a fruitful approach for explaining and understanding the observed behavior, as well as providing hypotheses for the underlying mechanisms.In this contribution we review mathematical and computational approaches to calcium signalling in plants. We present a systems biology dissection of calcium signalling processes with some selected examples that demonstrate how this approach has helped to unravel complex phenomena and guide further experiments. Whilst the review aims at being comprehensive it is not exhaustive and we apologise to those authors whose work is not adequately represented. A number of common methodological techniques are summarised and we provide key equations and list some popular software packages. The review concludes with an outlook of future challenges and application areas.2+ which has an effective ionic diameter of 2 Å. In seawater Ca2+ is the fifth most abundant dissolved ion by mass at around 400 mg/L or 10 mM. The Ca2+ concentration of drinking water varies from about 1 to 135 mg/L  of 100 nM a cell with a cytoplasmic volume of 200 μm3 would contain about 12,000 ions, a nuclear volume of 100 μm3 would have around 6,000 ions but a small organelle of 1 μm3 would only hold around 60 calcium ions. Given these numbers and the locality of calcium signatures, low copy number effects are likely to be relevant at physiological Ca2+ concentrations in smaller compartments  spiking in the cytosol. The model consists of a system of ODEs that describes calcium fluxes between the cytosol, the exterior and an indeterminate internal store. Since the vacuole is the largest calcium store it could be seen as a natural choice, but, perhaps counterintuitively, it is its size that excludes it. The clue to the store identity is given by the fact that at least two variables are required for self-sustained repetitive spiking  re reet al. . Mu. Mu2+ sp spiking , and the spiking . Blockin spiking  and gene spiking . All of  factors .vs. Myc pathway? These final two questions are discussed in a more general setting in the specificity section towards the end of this review.Key questions in this field are therefore: (1) how are the oscillations established and what are the molecular mechanisms of the core components; (2) what is the role of the nuclear membrane in generating peri-nuclear and nuclear oscillations; (3) how are the calcium oscillations decoded to affect downstream developmental programmes; and (4) is there a role for calcium in determining the specificity of the Nod et al.  showed the vacuolar concentration increasing during the day and the cytosolic concentration peaking sharply during the first few hours of darkness (as did the cytosolic-free [Ca2+]). The model predicted voltage and cytosolic-free [Ca2+] oscillations during the 3–4 h day to night transition that were noted to be broadly consistent with previous observations. The standout prediction, however, is the diurnal variation in the resting cytosolic-free [Ca2+], in particular the significant daytime increase despite the enhanced export of Ca2+ from the cytosol during this time. This counterintuitive prediction was explained as a result of plasma membrane hyperpolarisation, which limits the kinetics of the Ca2+-ATPase and Ca2+ channels located there. This mechanism provided an explanation without the requirement for a feedback loop, as suggested by Dodd et al.  by light. The relative importance of both nodes in this feedforward loop was estimated by frequency domain analysis (Bode plots). The hypothesis that rapid light input is associated with correct circadian timing was tested by subjecting over 3.5 thousand rhythmic transcripts from published microarray data to a Bode analysis. Over one thousand of these resulted in good models. The authors suggest that for the remaining transcripts their linear systems are not complex enough to capture the observed dynamic behavior.By using so-called hidden variables in the model it is possible to account for further complexities in the connections between variables, -fitting ,146. Optet al.  throughout the plant and they preceded stomatal closure. The authors noted a difference between repetitive Ca2+ oscillations seen in individual guard cells and the “spike-shoulder” pattern observed for a population of guard cells. They investigated whether low-temperature-induced [Ca2+]cyt increases measured from a population of guard cells were likely to represent the summation of cold-induced single-cell [Ca2+]cyt oscillations. In their model, the single-cell cytosolic Ca2+ concentration was represented by a modified sine-wave with peaks and troughs that decayed with time, and with the period treated as a normally-distributed random variable . The au2+ signals between cells. The excitation of a cell results in a calcium spike described by a sum of exponentials, and can be propagated to neighbour cells according to cellular automata type rules. Not surprisingly, the overall signal mostly differs from the individual cellular response when few cells are excited at the start, or there are many cells, or when it is difficult to transmit the excitation because of high thresholds for excitation. Most interestingly, this modelling approach permits the consideration of a great variety of complex geometries common in plants. The definition of the neighbours depends on the shape of the plant; certain geometries will lead to a successive stimulation of a cell at a time whereas others will lead to the simultaneous excitation of several neighbours at once. The architecture of delays determines how much information about the individual signal can be transmitted to the whole plant. To demonstrate, the author shows that when the morphology is elongated, branched or dendritic the shape of an individual spike is not recognisable at a global level.To study in more detail the mechanisms underlying the relation between the local and global signatures, Plieth  proposesMoreover, in those more complex shapes, the capacity of the first simulated cell to transmit its signature to the entire plant depends on its particular location on the plant. If this is true, we can conclude that the shape of the cell organelles influences the calcium signature. Therefore care must be taken during experimental measurements to avoid inferring from the global signature the local signal that a decoding protein is confronted with.2+ spiking responses are associated with different key stages of transcellular infection. Moreover, the efficacy of cell-to-cell signal transmission must necessarily depend on the cell compartment that initiates calcium signalling. In a symbiosis context, Ehrhardt et al. i to 600 nM in Commelina guard cells that had been injected with the fluorescent indicator dye fura-2 . It will be interesting to see whether the snapshot reporter concept proposed by Tsien [2+] when an external trigger  they are both measured using fluorescence microscopy. By accurate calibration of the signal-to-Caund Ca2+ . Furtherund Ca2+  each witby Tsien  for periuggested ) is applin vivo observations is likely to transform theoretical biology; a major limiting issue faced by modellers is the availability of experimentally quantified parameters for use in their models. The development of experimental processes that can help fill such gaps will be of huge benefit to the development of our understanding and will result in more accurate models. One such experimental technique that may be of great benefit to the field of calcium signalling is Fluorescence Correlation Spectroscopy (FCS)   ,203). FC. FCin vilmodulin . The teclmodulin ,206. Conlmodulin . With suA single fluorescent molecule may be about 1–2 nm in size but when imaged through a microscope this nanoscale source of light appears as a fuzzy spot with an intensity profile that defines the point spread function (PSF). If the distance that separates two identical sources of light is less than the distance of the PSF they appear as a single unresolved object. Even under optimal conditions the optical resolution of conventional fluorescence microscopy is limited to approximately half of the wavelength of the light used, which means that only objects separated by at least 200 to 350 nm can be resolved. This is the approximate size of intracellular organelles thus leaving fundamental biological processes beyond reach. Super-resolution techniques ,216,217 i.e., fluctuations due to low copy numbers, is beyond doubt. Likewise, the in vitro dilution ranges and relatives enzyme/ligand ratios for which the equations of enzyme kinetics were derived are far from physiological conditions. How diffusion varies locally in a crowded environment and how local changes in substrate and enzyme availability affect cellular processes is relatively poorly understood. In particular, for calcium signalling these local properties are likely to be of importance for both the encoding and decoding processes. It is not known how the physics of transport in temporally and spatially heterogeneous, crowded, viscous fluids might influence, for instance, channel gating, or other conformational changes. In the same way that concentrations become ill-defined for small compartments with low copy numbers, ionic currents, temperature and other macroscopic quantities that determine the behaviour of many models involving calcium may need reconsideration.With the increasing resolution of imaging technology and our enhanced understanding of processes within the cell, it is not impossible that a number of the basic assumptions underlying standard enzyme kinetics (and the models that use them) may need revisiting. The concept and validity of a concentration and its associated continuous change has already been shown to deviate from discrete models for small particle numbers and the importance of “stochasticity”, 3R channel is about 20 nm, with internal structures of the order of 2–3 nm [et al. [There is thus an enormous potential in these emerging experimental techniques to further the understanding of calcium signalling. The relevant components can only be imaged by super-resolution: e.g., the overall size of an IPf 2–3 nm , and thef 2–3 nm . Moreovef 2–3 nm ,229,230 f 2–3 nm ,234,235. [et al.  were abl


Cells use both extracellular and intracellular Ca2+ pools to modulate the intracellular Ca2+ concentration. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R), located on the endoplasmic reticulum (ER), is responsible for the release of Ca2+ from the intracellular store. The proteins STIM1 and STIM2 are also located on the ER and they are involved in the activation of a store-operated Ca2+ entry (SOCE). Due to their Ca2+ sensor property and their close proximity with IP3Rs on the ER, STIMs could modulate the activity of IP3R. In this study, we showed that STIM1 and STIM2 are expressed in bovine aortic endothelial cells and they both interact with IP3R. While STIM2 appears to play a minor role, STIM1 plays an important role in the regulation of agonist-induced Ca2+ mobilization in BAECs by a positive effect on both the SOCE and the IP3R-dependent Ca2+ release.The endothelium is actively involved in many functions of the cardiovascular system, such as the modulation of arterial pressure and the maintenance of blood flow. These functions require a great versatility of the intracellular Ca The versatility of Ca2+ signaling resides in the fact that different signals can be encoded spatio-temporally by varying the frequency and the amplitude of the Ca2+ response 2+ pools to modulate the intracellular Ca2+ concentration. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is responsible for the release of Ca2+ from the endoplasmic reticulum (ER), the main intracellular Ca2+ store by which the concentration of cytosolic Ca2+ is modulated 3R subtypes have been identified to date  and they associate into tetramers to form functional Ca2+ selective ligand-gated channels 3R is activated by signaling cascades that generate IP3. Briefly, an extracellular agonist binds to its specific receptor, which activates phospholipase C (PLC) via a G-protein or a tyrosine kinase activity. PLC then catalyzes the cleavage of phosphatidylinositol 4,5-bisphosphate into diacylglycerol and IP3, which diffuses into the cytosol and activates IP3R, its receptor/channel The endothelium is the monolayer of cells lining the interior of blood vessels. The endothelial cells that constitute this monolayer are actively involved in many functions of the cardiovascular system, including the regulation of immune responses, the adjustment of blood-tissue permeability, the repair of blood vessels, the modulation of arterial pressure, and the maintenance of blood flow 2+ level in the ER declines, a mechanism of Ca2+ entry through the plasma membrane is activated. This “store-operated Ca2+ entry” (SOCE) serves to sustain the Ca2+ response and to restore the Ca2+ level in the ER 2+ sensors that, at low luminal Ca2+ concentration, activate plasma membrane Ca2+ channels members of the Orai or TRPC families As the Cain vitro VEGF-induced tubulogenesis 3R-dependent Ca2+ release and SOCE activity both contribute to shape the agonist-induced Ca2+ response 2+ in the ER, that they also control the activity of Ca2+ channels and that they are located in the ER, where IP3Rs also are, make them good candidates to modulate the IP3R activity. However, little attention has been given to the potential role of STIMs on IP3R-dependent Ca2+ release. In this study, we showed that STIM1 and STIM2 are expressed in bovine aortic endothelial cells (BAEC) and participate to SOCE. Most importantly, we showed that STIMs interact with IP3R-1 and that the knockdown of STIM1, but not that of STIM2, dampens the IP3R-dependent Ca2+ release in BAECs.In endothelial cells, STIM1 has been identified as a crucial component of SOCE and consequently, it is involved in specialized functions that depend on SOCE such as NO production, cell proliferation and 3R-1 antibody was from ABR Affinity Bioreagents . Anti-STIM1 antibody was from Millipore  and anti-STIM2 antibody was from Abcam . Protein AG-Agarose beads and anti-HA probe antibody were from Santa Cruz Technology . siRNAs against bovine STIM1 and STIM2, and siRNA control #3 were from Dharmacon, Inc. . LipofectAMINE 2000 transfection reagent was from Invitrogen . ATP and BK were from Sigma-Aldrich .Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin-glutamine were from Gibco Life Technologies . Fura-2/AM was from Calbiochem . Anti-IP2. They were used between the 5th and 20th passages. Experiments were approved by the “Comité facultaire de protection des animaux” of the Faculty of Medicine and Health Sciences of the Université de Sherbrooke.Bovine thoracic aortas from calves 8–10 months of age were obtained from a nearby slaughterhouse . BAECs were isolated and characterized as previously described 2PO4, 8 mM Na2HPO4, pH 7.4) and solubilized for 30 min on ice in lysis buffer . The lysates were clarified by centrifugation at 10 000× g for 10 min. For the immunoprecipitation studies, identical amounts of protein from each sample were incubated overnight at 4°C with 5 µg/ml of a specific antibody. The immune complexes were collected by incubating the mixtures with 50 µl (50% suspension) of protein A/G-agarose beads. Nonspecifically bound proteins were removed by washing the beads three times with 1 ml of lysis buffer, and bound material was solubilized in 50 µl of 2× Laemmli sample buffer Cells were washed twice with phosphate-buffered saline  prepared in PBS. After three washes with PBS, cells were incubated for 1 h at room temperature with secondary AlexaFluor 488-conjugated goat antibodies against rabbit IgG (1∶300 dilution) or AlexaFluor 594-conjugated goat antibodies against mouse IgG (1∶300 dilution) . After extensive washing with PBS, cover glasses were mounted on microscope slides using Vectashield  and examined on a Zeiss Axio Observer microscope (40× oil immersion objective). Pictures were obtained with a Zeiss Axiocam MRm camera using AxioVision LE software. In control experiments performed in parallel, no specific immunofluorescent staining was observed when primary antibodies were omitted.Endothelial cells were seeded on 25 mm cover glasses in 6-wells plates and maintained in culture until they reached 60% confluence. Cells were washed with PBS and fixed with 100% methanol for 10 min at −20°C. Non-specific sites were blocked with 2% BSA in PBS for 1 h at room temperature. After being washed, cells were incubated overnight at 4°C with primary anti-STIM1 and anti-IP5′-CCAAGGAGCACAUGAAGAAdTdT-3′ and 5′-GGUUCCUCGUGUACUUCUUdTdT-3′; and those against STIM2 are 5′-UGACAAAGAUGGUGGAAUCUUdTdT-3′ and 5′-UUACUGUUUCUACCACCUUAGdTdT-3′. Fresh medium was added 5 h post-transfection, and experiments were conducted 48 h after transfection.Six-well plates of BAECs were cultured to 70% of confluence. BAECs were transfected with 40 nM of siRNA using 0.2% of LipofectAMINE 2000 following the protocol provided by the manufacturer (Invitrogen). The cells were maintained in DMEM 10% FBS without antibiotics. The sequences of the sense and anti-sense small interfering RNAs against STIM1 are Total RNA was extracted from BAECs, transfected or not with appropriate siRNA oligonucleotide, using TRIzol. RNA quality and presence of contaminating genomic DNA was verified as previously described 4, 5 mM glucose, 1.8 mM CaCl2, 20 mM Hepes, pH 7.4) and loaded with 0.4 µM fura 2-AM for 30 min at room temperature in the dark. The cells were then washed and bathed in fresh HBSS for 30 min to ensure complete hydrolysis of the fura-2/AM before placing the coverslip in a circular open-bottom chamber mounted on the stage of a Olympus IX71 microscope  equipped with a Lambda DG-4 illuminator . Fluorescence from isolated fura-2-loaded cells was monitored by videomicroscopy using 340 nm and 387 nm excitatory wavelengths, and emitted fluorescence was recorded at 510 nm. Images were digitized and analyzed with the MetaFluor software . The data are expressed as the intracellular free Ca2+ concentration (nM) calculated from the 340/387 fluorescence ratio according to BAECs grown on glass coverslips were washed twice with HBSS . The concentration response curve fitting were done using the R statistical software 2.14.1  and the EC50s and maximal responses were expressed as best fit ± standard error (SE).All experiments were performed at least three times. 3-sensitive Ca2+ pool content and the activation of SOCE were evaluated in intact BAECs. BAECs were bathed in a nominally Ca2+ free medium and treated with 1 µM thapsigargin (a SERCA pump inhibitor). Thapsigargin increased the intracellular Ca2+ to a similar level in BAECs transfected with siCtrl, siSTIM1 or siSTIM2  or strongly (for STIM1) affected their SOCE activity.The qPCR analysis shown in  siSTIM2 . The ave siSTIM2 , left. Th siCtrl . The aveh siCtrl , right. 3R under basal conditions, we first examined if their intracellular localization made this possible in BAECs. 3R-1 antibodies in untransfected and unstimulated BAECs. Using the anti-STIM1 antibody, the fluorescence was widely distributed throughout the cell with a higher intensity in the perinuclear region corresponding to the endoplasmic reticulum. The outer limits of the cell were not clearly defined, which indicates that the plasma membrane was not stained (left panel). Similar results were obtained with the anti-IP3R-1 antibody . The overlay image of the two staining clearly shows that STIM1 and IP3R-1 were mostly present in the same region of the endoplasmic reticulum and that their physical interaction was possible in a wide part of the cell (right panel). A co-immunoprecipitation approach was used to further verify whether these two proteins interact together. Isoform specific antibodies were used to precipitate the IP3R-1 from BAECs lysates and the presence of STIM1 and STIM2 in the resulting immune complex was verified with isoform specific antibodies.To verify whether STIMs could functionally interact with IP3R-1 , two well known Ca2+-mobilizing agonists in BAECs 3R-dependent Ca2+ release, the experiments were done in a nominally free Ca2+ extracellular medium. 2+ responses of pre-selected BAECs (average response of 25 cells) transfected with siCtrl (solid black line), siSTIM1 (dashed black line) or siSTIM2 (solid gray line) after stimulation with 100 nM ATP, a submaximal concentration to release Ca2+. ATP (100 nM) increased the intracellular Ca2+ concentration from around 40 nM to 180 nM in cells transfected with siCtrl, to 125 nM in cells transfected with siSTIM1 and to 171 nM in cells transfected with siSTIM2. A submaximal concentration of BK (5 nM) increased the intracellular Ca2+ concentration from around 40 nM to 160 nM in cells transfected with siCtrl, to 106 nM in cells transfected with siSTIM1 and to 147 nM in cells transfected with siSTIM2 . The peak Ca2+ response obtained with a maximal concentration of ATP was 206±5 nM Ca2+ in cells transfected with siCtrl, 205±5 nM Ca2+ in cells transfected with siSTIM2 and 138±4 nM Ca2+ (67% of control response) in cells transfected with siSTIM1.We repeated these experiments using increasing concentrations of ATP and reported graphically the mean peak amplitude obtained with each concentration, as shown in 2+ response upon stimulation with high concentrations of BK. The peak Ca2+ response obtained with a maximal concentration of BK was 131±4 nM Ca2+ in cells transfected with siCtrl, 129±6 nM Ca2+ in cells transfected with siSTIM2 and 80±5 nM Ca2+ (62% of control response) in cells transfected with siSTIM1. These results also show that BK is less efficient than ATP to mobilize Ca2+. Indeed, in control cells, the maximal response obtained with BK corresponds to only 64% of the maximal response obtained with ATP. Interestingly, while the maximal response obtained with BK is 36% lower than that obtained with ATP, the reduction of the maximal response of cells transfected with siSTIM1 is similar with both hormones (about 35% lower than that of control cells). To illustrate the effect of the knockdown of STIM1 and STIM2 on the apparent affinities of both agonists, the data shown in 3R-dependent agonist-induced intracellular Ca2+ release in BAECs without affecting the apparent affinity of the Ca2+-mobilizing agonists.Concentration-response curves were also obtained using BK . As obse3R-dependent Ca2+ release and SOCE contribute to shape the agonist-induced Ca2+ response 2+ entry 3R-dependent Ca2+ release. We showed that STIM1 and STIM2 are expressed in BAECs, which is also the case in most cellular types including endothelial cells 2+ available in the ER, the knockdown of STIM1 nearly abolished the SOCE while the knockdown of STIM2 resulted in a minor reduction of the SOCE. A strong abolition of the SOCE induced by the knockdown of STIM1 has also been reported in human umbilical vein endothelial cells (HUVEC and EA.hy926 cells), in porcine aortic endothelial cells (PAEC) and in mouse lung endothelial cells, thus confirming the crucial role of STIM1 in endothelial SOCE 2+ concentration in different cell types including HUVEC 2+ concentration was lowered by about 3 nM after silencing STIM2 2+ level after silencing STIM1 or STIM2.In endothelial cells, both IP3R is known to be finely tuned by a variety of interacting molecules and by the luminal or cytosolic Ca2+ concentration 2+ in the ER and the basal cytosolic Ca2+ concentration did not change when STIM1 or STIM2 were silenced, the only way they can modulate the IP3R-dependent Ca2+ release is via an interaction with a component that leads directly to Ca2+ release via IP3R.The activity of IP3R-1 and STIM1 share a similar pattern of distribution throughout BAECs, which is a sign of proximity between them. The wide distribution of these two proteins could allow the regulation of the Ca2+ release at a cellular scale rather than only in focalized regions of the cells. Using co-immunoprecipitation studies, we demonstrated that STIM1 and STIM2 interact with IP3R-1. Another example of proximity between STIMs and IP3Rs has been provided in HEK293 cells where the formation of a protein complex composed of Orai1, STIM1, TRPC3, RACK3 and IP3R-1 is formed upon activation with ATP or Carbachol 3R-3 has been observed upon activation with ATP 3R-mediated Ca2+ release are also more frequently observed 3R.We showed that IP3R-dependent intracellular Ca2+ release induced by ATP and BK in intact BAECs. Similar results were observed in HEK293 cells where the formation of a protein complex composed of Orai1, STIM1, TRPC3, RACK3 and IP3R-1 was shown to increase the IP3R-dependent Ca2+ release 2 showed a reduced intracellular Ca2+ release 2+ release via IP3R.Our results also revealed that the knockdown of STIM1, but not that of STIM2, dampens the IP3R-dependent intracellular Ca2+ release in BAECs stimulated with ATP or BK without affecting their apparent affinity. These results suggest that STIM1 exerts a direct effect on IP3R functionality. Recently, Santoso et al. 3R-3 to prevent the binding of polycystin-2 in MDCK cells. Located on the ER, polycystin-2 is a Ca2+ channel activated by Ca2+ that enhances the IP3-dependent Ca2+ release when interacting with the IP3R 3Rs by other molecules. An example of such a molecule is CIB1, a widely expressed Ca2+ binding protein, that has been shown to bind and inactivate IP3R, thus reducing the number of channels available for activation by IP33R activity by stabilizing a conformation allowing an increased flux of Ca2+ upon activation. Some interacting proteins or kinases have been shown to directly modulate IP3R gating by affecting its open probability (for a review see 2+ signal necessary to promote HUVEC migration 3R activity is compatible with such a mechanism. Further investigations are needed to establish precisely how STIM1 induces a positive effect on IP3R functionality in endothelial cells.Using concentration-response analyses, we demonstrated that the knockdown of STIM1, but not that of STIM2, reduced the maximal IP3R-1, which is a sign of proximity between STIMs and IP3R populations. Moreover, we demonstrated that STIM1, but not STIM2, is a positive regulator of IP3R in BAECs. The mechanism does not involve a change in the sensitivity of IP3R for IP3, but the results rather suggest that STIM1 increases the efficacy of IP3R. Therefore, while the role of STIM2 appears to be minor, STIM1 plays an important role in the regulation of agonist-induced Ca2+ mobilization in BAECs by a positive effect on both the SOCE and the IP3R-dependent Ca2+ release.In conclusion, we showed that STIM1 and STIM2 are expressed in BAECs and that SOCE strongly depends on STIM1 and partially on STIM2. We also identified STIM1 and STIM2 as interacting partners for IP


Bone marrow-derived mesenchymal stromal cells (BM-MSCs) play a fundamental role in the BM microenvironment (BME) and abnormalities of these cells may contribute to acute myeloid leukemia (AML) pathogenesis. The aim of the study was to characterize the cytokine and gene expression profile, immunophenotype and cytogenetics of BM-MSCs from AML patients compared to normal BM-MSCs from healthy donors. AML BM-MSCs showed decreased monocyte chemoattractant protein-1 levels compared to normal BM-MSCs. AML BM-MSCs expressed similar β1 integrin, CD44, CD73, CD90 and E-cadherin compared to normal BM-MSCs. Cytogenetic analysis revealed chromosomal aberrations in AML BM-MSCs, some overlapping with and others distinct from their corresponding AML blasts. No significant difference in gene expression was detected between AML BM-MSCs compared to normal BM-MSCs; however, comparing the differences between AML and MSCs from AML patients with the differences between normal hematopoietic cells and normal MSCs by Ingenuity pathway analysis showed key distinctions of the AML setting: (1) upstream gene regulation by transforming growth factor beta 1, tumor necrosis factor, tissue transglutaminase 2, CCAAT/enhancer binding protein alpha and SWItch/Sucrose NonFermentable related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4; (2) integrin and interleukin 8 signaling as overrepresented canonical pathways; and (3) upregulation of transcription factors FBJ murine osteosarcoma viral oncogene homolog and v-myb avian myeloblastosis viral oncogene homolog. Thus, phenotypic abnormalities of AML BM-MSCs highlight a dysfunctional BME that may impact AML survival and proliferation. For example, growth factors from stromal cell lines confer resistance to targeted therapies and chemotherapy in melanoma, glioblastoma and colorectal cancer cell lines in a co-culture system.8 Chemotherapy-induced damage of benign prostatic stromal cells resulted in diminished chemotherapy effect and promoted prostate cancer cell survival and progression.9 In breast cancer, tumor cell gene expression changed when co-cultured on different tissue microenvironments.10 In a study of surgically resected hepatocellular carcinoma, the gene expression profile of the non-malignant peri-tumoral tissue in the resection specimen could predict disease relapse, whereas the expression profile of the malignant cells themselves did not.11 These findings suggest that alteration in the surrounding stromal tissue independently contributed to disease behavior. Work in mouse models have provided evidence that alterations in the BME can contribute to the evolution of hematologic malignancy: for example, knockout of the RNase III endonuclease Dicer1 specifically in mesenchymal osteoprogenitor cells resulted in myelodysplasia and emergence of AML despite maintaining genetic integrity in the hematopoietic lineage.12 Another study showed that an activating mutation of β-catenin in osteoblasts induced AML through Notch signaling.13 Considering these reports of stroma-microenvironment aberrations specifically influencing the evolution of malignant conditions, we hypothesized that the BM-derived MSCs (BM-MSCs) from AML patients would exhibit distinct genotypic and phenotypic differences compared to BM-MSCs from normal healthy donors.Acute myeloid leukemia (AML) is a heterogeneous disorder that arises in the bone marrow microenvironment (BME) under the influence of mesenchymal stromal cells (MSCs), endothelial cells, osteocytes, pericytes, adipocytes, monocytes, fibroblasts and their secreted extracellular glycosaminoglycans, chemokines and cytokines. The BME plays a fundamental role in the growth, proliferation and survival of AML cells. Leukemia cell growth in the BME also disrupts normal hematopoiesis.14 Cytogenetic abnormalities have been reported in BM-MSCs from patients with myelodysplastic syndrome (MDS) and AML, which were distinct from leukemic blasts and may be associated with inferior outcomes.15 A distinctive gene expression profile of MSCs from pediatric cases of MDS and AML was found compared with healthy donors.16 To gain further insight into the characteristics of MSCs in AML, we aimed to characterize the cytokine and gene expression profile, immunophenotype and cytogenetics of primary BM-MSCs isolated from AML patients in comparison to healthy donors.The central component of the BME are MSCs, which are capable of giving rise to different cell lineages, such as osteoblasts, adipocytes and chondroblasts.The study was conducted with written informed consent in accordance with the Declaration of Helsinki and under the guidelines of the University of Washington/Fred Hutchinson Cancer Research Center (FHCRC) Cancer Consortium Institutional Review Board, which approved the study.MSC cultures were successfully derived from BM aspirates of 22 AML patients and 5 healthy donors, either from fresh or cryopreserved samples. Mononuclear cells (MNCs) were isolated from BM samples using lymphocyte separation medium . Approximately, 2 to 5 million fresh or cryopreserved BM cells were cultured overnight in Iscove's Modified Dulbecco's Media , 15% heat-inactivated fetal bovine serum, 15% horse serum, 1% penicillin–streptomycin , then the media was replaced (removing the non-adherent cells) over the adherent cells by non-hematopoietic expansion media  in 5% oxygen within a hypoxic chamber to more closely approximate the sinusoidal regions of the BM. Cells were also cultured in normal 21% oxygen conditions for comparison of cytokine production at different oxygen concentrations. After 48 h, non-adherent cells were removed from the culture by washing with phosphate-buffered solution. Over a period of 1–2 weeks, adherent, spindle-shaped cells were derived and subcultured weekly by trypsinization for ~10–15 passages. The MSCs were collected for analysis after the third to fourth passages. Similar cultures were derived from normal BM from healthy donors and normal homo sapiens (HS)-5 and HS-27a stromal cell lines  were also used for comparison.Flow cytometry was performed on single stromal cell suspensions that were stained with the following anti-human monoclonal antibodies: allophycocyanin-conjugated antibody against CD90, CD34 or CD45, fluorescein isothiocyanate-conjugated antibody against CD324 or CD146, R-phycoerythrin-conjugated antibody against CD14, CD29, CD44 or CD73, and PerCP Cy5-5-conjugated antibody against CD105 to confirm their mesenchymal origin.Conditioned media was collected from adherent, subconfluent MSC cultures (5 and 21% oxygen) after cell components were removed by centrifugation. The Luminex method  was used for detection of the following cytokines: granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte CSF (GM-CSF), macrophage CSF, stem cell factor, interleukin 6 (IL-6), IL-12, monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor alpha (TNF-α). Enzyme-linked immunosorbent assay was used for detection of stromal-derived factor-1. All assays were performed at the FHCRC Immunoassays Core Facility according to manufacturer's instructions.17 Fluorescence in situ hybridization was performed on one sample using a centromere region of chromosome 8p11.1-q111.1 probe . Slides were enumerated using AxioImager Z1/2 microscopes  on 600 interphase nuclei. Clonal results above the laboratory-established false-positive cutoff values were considered positive.Cells actively growing in logarithmic phase were incubated with 0.1 μg/ml colcemid for 60 min at 37 °C to arrest cells in metaphase. Cells were then treated with the pre-warmed hypotonic solution (0.075 M KCl) for 30 min before fixation in methanol and glacial acetic acid (2.5:1 ratio). Fixed cells were dropped on glass slides, treated with 0.025% trypsin in 0.9% NaCl and stained with 1:4 diluted Wright's stain (pH 6.8) for Giemsa-Trypsin-Wright banding. Metaphases were analyzed under a microscope at a magnification of × 1250 for chromosome count and structural integrity. At least 20 metaphases were analyzed for each sample. Karyotypes were written according to current International System for Human Cytogenetic Nomenclature (ISCN 2013).http://www.bioconductor.org/) and log-2 transformed. Significant differential gene expression was defined as |log2(ratio)|>0.585 (±1.5-fold) and false discovery rate of 5%. Probe sets retained after application of signal intensity and variance filters were used in a principal component analysis18 to generate a map of AML BM-MSCs, AML blasts, normal BM-MSCs, normal hematopoietic MNCs and the stromal cell lines (HS-5 and HS-27a) by projection of the data onto the first two principal components . These probe sets were also used to generate a heat map . These values were hierarchically clustered by probe.For all patients and cell lines, 100 ng of high-quality RNA was used. Total RNA was extracted using RNeasy mini kit , labeled and hybridized to Illumina's HumanHT Expression BeadChip microarrays according to the manufacturer's instructions by the FHCRC Genomics Core Facility. Signal intensities were quantile normalized using the ‘lumen package' from Bioconductor . IPA determines biologically enriched pathways and functions based on direct and indirect relationships in published literature. The P-value is calculated using a right-sided Fisher's exact test and measures the statistical significance of a particular function or pathway in our data with respect to the reference set defined by IPA. A key feature of IPA is the upstream regulator analysis, which identifies putative upstream regulators that are activated or inhibited in our gene expression data sets. Upstream regulators include transcription factors, cytokines, microRNAs, receptors, kinases, chemicals and drugs. Activation or inhibition is assigned based on relationships derived from the literature and various databases and the direction of expression change in the array data provided. It also identifies candidates that are statistically enriched when the direction cannot be ascertained. The P-value is calculated using Fisher's exact test.GenePlus software , which uses estimating equation techniques was also used to determine differential expression between groups using analysis of variance to calculate 2) were made using the Student's t-test and the two-sided Fisher's exact test (dichotomous variables). P-value <0.05 was considered statistically significant.Results in cytokine levels are the average of experiments carried out in duplicate. Comparisons in cytokine levels between different groups  and different oxygen conditions (5 vs 21% OThe spindle-shaped cells grew adherent to the culture dish and became subconfluent or confluent by 1–2 weeks. The cells were fibroblast like in morphology with a few long processes and large nuclei . The MSC1 integrin (CD29), CD44, CD73, CD90 and E-cadherin (CD324). The normal BM stromal cell lines, HS-5  as compared to normal BM-MSCs from healthy donors (approximatly five times lower). We observed a trend toward reduced GM-CSF and IL-6 levels produced by AML BM-MSCs compared with BM-MSCs from healthy donors (P>0.07). In hypoxic conditions as compared to normoxic conditions, however, there was a strong reduction in cytokine production in AML BM-MSCs with significantly lower levels of GM-CSF, stem cell factor and TNF-α (P<0.04), and a trend toward lower levels of MCP-1  and found no statistically significant differences in the levels of eight of the nine cytokines when comparing AML BM-MSCs to normal BM-MSCs . One not P=0.09; . Tables P-values 4.81 × 10−21 to 1.26 × 10−38), with the predicted activation states of TGFβ1, TNF, TGM2 inhibited; and (2) hepatic fibrosis/hepatic stellate cell activation, integrin, leukocyte extravasation, IL-8 and axonal guidance signaling as overrepresented canonical pathways . IPA analysis was used to identify putative regulatory, functional or pathway-specific gene expression differences between AML BM-MSCs and AML blasts compared with the differences in gene expression between normal BM-MSCs and normal hematopoietic MNCs from healthy donors. The results of this analysis showed key distinctions in the leukemia-derived population to be in: (1) upstream regulation of gene expression by transforming growth factor beta 1 (TGFβ1), TNF, tissue transglutaminase 2 (TGM2), CCAAT/enhancer binding protein alpha (C/EBPA) and SWItch/Sucrose NonFermentable (SWI/SNF) related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (SMARCA4)  in patient AML 073 and der, −6, del(7)(q22q34), +8, −18, −20 and −22 in patient AML 094—as well as distinct aberrations. Thus, BM-MSCs in a small sample of AML patients demonstrate abnormal cytogenetics, some overlapping with and others distinct, from the cytogenetic aberrations present in their corresponding leukemic blasts.Given the cited murine model data showing how directed mutations of stromal cells can lead to myelodysplasia and emergence of AML,19 MCP-1 is secreted by both AML blasts and stromal cells, and serum levels are increased in AML patients and appear to vary depending on AML subtype.20 Our data show that MCP-1, GM-CSF, stem cell factor and TNF-α levels produced by AML BM-MSCs are vastly different depending on oxygen levels and are much lower in hypoxic conditions similar to the sinusoidal regions of the BM. We observed a trend toward reduced GM-CSF and IL-6 production from AML BM-MSCs compared to normal BM-MSCs. The lower GM-CSF levels observed would be consistent with the view that the leukemic BME results in a diminished ability to support normal hematopoiesis.AML arises from the clonal evolution of a hematopoietic progenitor cell that acquires successive genetic alterations. The mechanisms by which MSCs influence AML development and progression are under active investigation. We found significantly reduced MCP-1 levels from BM-MSCs from AML patients compared to BM-MSCs from healthy donors. MCP-1 is one of the major chemokines that regulate migration and infiltration of monocytes and macrophages. Apart from recruiting and directing leukocyte movement, MCP-1 also exhibits anti-tumor activity, in promoting Fas cell surface death receptor-ligand-mediated apoptosis in cultured endometrial stromal cells.21 and plasma-derived exosome content of TGFβ1 from AML blasts may predict AML relapse.22 TNFs have been shown to differentially affect cytokine expression and migration properties of MSCs.23 The pro-inflammatory effects of TNF increase AML survival and proliferation by upregulation of anti-apoptotic genes.24 Tissue transglutaminase 2 (TGM2) expression is elevated in AML along with many other cancer cell types and increased gene expression correlates with increased expression of adhesion and apoptosis-regulating proteins, such as fibronectin, integrin β3 and focal adhesion kinase.25 TGM2 expression is induced by TGFβ, TNF-α and IL-6, and evidence supports its common role in chronic inflammation and cancer.26 The C/EBPA gene belongs to the CCAAT/enhancer-binding protein family of transcription factors, which is a master regulator of hematopoietic cell proliferation and differentiation and is frequently mutated in AML. Double mutations in the C/EBPA gene confers a relatively favorable prognosis in AML.27 SMARCA4 is a catalytic subunit of the SWI/SNF (brahma related gene 1 (BRG1) associated factor) chromatin regulatory complex, which is frequently mutated in cancer and has recently been shown to be critical for AML maintenance and proliferation.29 Integrin β3-mediated signaling has been shown to be required for leukemia development in a xenotransplantation study.30 IL-8 is a pro-angiogenic and chemotactic cytokine, whose levels are increased in AML cells when co-cultured with fibroblasts and stromal cells, which likely contributes to the increased vessel density in AML BM and thus important in leukemogenesis.31We observed significant differences in gene expression in AML BM-MSCs compared to leukemic blasts, but not compared to BM-MSCs from healthy donors. IPA identified the regulatory pathways involving the genes that distinguished leukemia cells from their stroma (AML blasts vs AML MSCs) and those that distinguished normal hematopoietic cells from their stroma  to be: (1) TGFβ1, TNF, TGM2, C/EBPA and SMARCA4 as upstream regulators of gene expression; (2) integrin and IL-8 signaling as overrepresented canonical pathways; and (3) upregulation of the transcription factors FOS and MYB. TGFβ1 has been shown to differentially regulate myeloid leukemia in the BME with suppressive effects on CML leukemia stem cells and proliferative effects on AML stem cells,The IPA revealed upregulation of the transcription factors FOS and MYB, which regulate cell proliferation, differentiation and transformation. FOS, one the upregulated molecules in AML, forms a complex that regulates TGF-β signaling. A downstream target of TGFβ1 is MCP-1. Thus, the diminished MCP-1 production from AML BM-MSCs may be the result of FOS-mediated inhibition of TGF-β signaling. The IPA reveals that leukemic cells (AML BM-MSCs and AML blasts) show significant differences in upstream gene regulation and canonical pathway signaling, which results in a complex interplay of upregulation and downregulation of signaling molecules compared to normal cells , and highlights potentially the most important pathways and molecules to target as biomarkers or for combinatorial therapeutic manipulation.32 In contrast to the present study, we did not identify any significant differences gene expression in the BM-MSCs from AML patients compared to the BM-MSCs of healthy donors. This discrepancy could in part be explained by the age difference in the study population, with the previous study involving pediatric cases of MDS/AML. The BME undergoes changes with aging, such as increased adipogenesis and decreased osteoblastogenesis, and an aged BM may facilitate better expansion of leukemic cells compared to a young BM.33 In addition, the length of time in culture, number of passages and tissue culture media could explain the differences in findings. A study by Jacamo et al.34 recently showed that primary BM-MSCs from leukemia patients expressed nuclear factor kappa beta target genes at higher levels than their normal BM-MSC, both upon co-culture with leukemic cells and in monoculture. In their study, the BM-MSCs were in culture for 4 to 8 weeks before genetic analysis. We did not detect differences in our AML BM-MSCs compared to normal BM-MSCs from healthy donor in gene expression profiling in monoculture at early passages.A previous study showed a distinctive gene expression profile of MSCs of MDS and AML patients compared with MSCs of healthy donors using microarray analysis.et al.35 showed no significant functional differences between MDS-derived BM-MSCs and normal BM-MSCs, with similar expression of cell adhesion and extracellular matrix proteins, ability to differentiate and support hematopoiesis in vitro, even among stromal cells with severe chromosomal abnormalities. Another group, however, showed decreased MSC plasticity and hematopoietic supportive capacity in higher-risk MDS patients,36 suggesting that there may be heterogeneity. In support of previous studies, we also observed that BM-MSCs from AML patients expressed similar cell-surface markers and adhesion proteins compared to BM-MSCs from healthy donors, such as β1 integrin, CD44, CD73, CD90, and E-cadherin.A study by Flores-Figueroa E 38 and one group has described aberrations in BM-MSCs in adult AML39 that were distinct from the leukemic cells. To the best of our knowledge, our study represents the first report of AML BM-MSCs sharing some similar chromosomal aberrations with their corresponding leukemic blasts. Whether chromosomal aberrations lead to functional alterations in BM-MSCs and how this influences disease progression and outcome in leukemia patients remain to be fully elucidated. In the report by Blau et al.,15 the presence of chromosomal aberrations in BM-MSCs was associated with inferior overall survival and disease-free survival. However, on multivariate analysis, cytogenetic changes in BM-MSCs were not an independent prognostic factor when the hematopoietic cell karyotype was included as a variable. We did not evaluate the relationship of BM-MSC aberrations to prognosis in our study due to the small sample size.We found BM-MSCs from AML patients demonstrated cytogenetic aberrations either distinct from or overlapping with their corresponding leukemic blasts. Several groups have demonstrated cytogenetic aberrations in BM-MSCs from MDS patients,In summary, we found reduced MCP-1 levels from primary BM-MSCs from AML patients compared to normal BM-MSCs from healthy donors and that cytokine secretion from AML BM-MSCs vary depending upon oxygen conditions, with much lower levels in hypoxic conditions that approximate the sinusoidal regions of the BME. The surface antigen and gene expression profile did not differ between BM-MSCs from AML patients compared to normal BM-MSCs. We found a few cases in which BM-MSCs from AML patients had overlapping and distinct cytogenetic aberrations with their corresponding leukemic cells. It will be of interest to evaluate the effect of manipulating MCP-1 levels in AML in future studies and to further characterize BM-MSCs with abnormal cytogenetics and the influence of reduced cytokine production in a hypoxic BME on leukemia stem cell quiescence.


The neonatal SERCA1b is the major Ca2+ pump in myotubes and young muscle fibers. To understand its role during skeletal muscle differentiation its synthesis has been interfered with specific shRNA sequence. Stably transfected clones showing significantly decreased SERCA1b expression (cloneC1) were selected for experiments. The expression of the regulatory proteins of skeletal muscle differentiation was examined either by Western-blot at the protein level for MyoD, STIM1, calsequestrin (CSQ), and calcineurin (CaN) or by RT-PCR for myostatin and MCIP1.4. Quantitative analysis revealed significant alterations in CSQ, STIM1, and CaN expression in cloneC1 as compared to control cells. To examine the functional consequences of the decreased expression of SERCA1b, repeated Ca2+-transients were evoked by applications of 120 mM KCl. The significantly higher [Ca2+]i measured at the 20th and 40th seconds after the beginning of KCl application  indicated a decreased Ca2+-uptake capability which was quantified by extracting the maximal pump rate . Furthermore, the rate of calcium release from the SR (610±60 vs. 377±64 μM/s) and the amount of calcium released (843±75 μM vs. 576±80 μM) were also significantly suppressed. These changes were also accompanied by a reduced activity of CaN in cells with decreased SERCA1b. In parallel, cloneC1 cells showed inhibited cell proliferation and decreased myotube nuclear numbers. Moreover, while cyclosporineA treatment suppressed the proliferation of parental cultures it had no effect on cloneC1 cells. SERCA1b is thus considered to play an essential role in the regulation of [Ca2+]i and its ab ovo gene silencing results in decreased skeletal muscle differentiation.The sarcoplasmic/endoplasmic reticulum Ca There are proteins in the SR specialized for this activity; the main players being the ryanodine receptor (RyR) through which Ca2+ is released into the sarcoplasm, the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) that reuptakes Ca2+ into the SR from the sarcoplasm, and calsequestrin (CSQ) that binds stored Ca2+ in the SR lumen. The three main SR proteins are expressed in developmental isoforms in fetal/postnatal stages and in myotubes of mammals. RyR expressed as RyR3  was calculated from the ratio of measured fluorescence intensities (R = F340/F380) as described by Grynkiewicz and colleagues i was calculated from the ratio of fluorescence intensities (R = F340/F380) using an in vivo calibration . To determine the activity of the Ca2+ pump and the Ca2+ flux entering the myoplasmic space, the Ca2+ binding to intracellular binding sites and the removal of Ca2+ from the intracellular space was modeled as presented in earlier reports i before the transients (108±4 nM in scrambled shRNA transfected and 109±4 nM in cloneC1 myotubes). However, following the KCl-evoked transients [Ca2+]i declined slower and returned to a significantly higher level in the clone C1 myotubes  indicating a decreased Ca2+-uptake capability of the SERCA pumps  the amplitude of the calcium transients in control and shRNA transfected cells were essentially identical  were suppressed significantly (p<0.01) as a result of SERCA1b silencing. Furthermore, the calculated integral for the releasable calcium . These observations suggest that the Ca2+-content of the SR and, consequently, the rate of Ca2+-release into the cytosol was decreased when the SERCA1b expression was interfered with    (for rawp<0.001) .th day after exchanging the proliferating medium to differentiating medium which corresponded to the terminal stage of differentiation. The diameter of terminally differentiated cloneC1 myotubes did not differ significantly (p>0.2) from that of the control  in the cloneC1 cultures, as compared to parental cells . On the other hand, CSA did not further decrease the rate of proliferation in cloneC1 cultures  indicating that the reduced proliferation seen in these cells was likely to be the consequence of their suppressed CaN activity  was successfully silenced in several clones of the mouse myogenic cells stably transfected with shRNA expressing vector. Because of the close homology between mouse and rat the sequence of shRNA was the same as the one which induced growth stimulation during the regeneration of rat soleus muscle after being transfected by a plasmid in a previous study i to its resting value following the KCl-evoked transients and the maximal transport rate of the Ca2+ pump (PVmax) were compared. To examine the Ca2+ content of the SR Ca2+ store the cells were treated with CPA.Ca(XLSX)Click here for additional data file.S3 Raw DataCell proliferation was characterized by the increase in the number of myogenic nuclei normalized to the value obtained after 24 hours of culturing.(XLSX)Click here for additional data file.S4 Raw Datath day of differentiation were fixed, and the number of DAPI-stained nuclei were counted manually, the diameter was measured by Image Browser.Cultures on the 5(XLSX)Click here for additional data file.S5 Raw DataSix separate wells were used from every single experimental group.(XLSX)Click here for additional data file.


We apply this method to investigate calcium clearance mechanisms in isolated magnocellular neurons of the rat supraoptic nucleus.Current optical methods based on fluorescent indicators permit to measure the intracellular calcium concentration with a high temporal resolution. To analyze the physiological mechanisms that underly the calcium dynamics, however, knowledge of the calcium fluxes into and out of the cell is needed. Here we present a method that permits to separately estimate the influx and clearance rates, based on the measurement of Ca2+ concentration during the transient is governed by a time-dependent influx Jinflux(t) through voltage-gated Calcium channels and by an outflux Jclearance that depends only on the instantaneous calcium concentration [Ca2+](t):In the simplest case, we assume that the cytoplasmic CaJclearance([Ca2+]). Near the end of the transient, Jclearance dominates over Jinflux, and Jclearance may be obtained directly as the measured Ca2+ decay rate [Jclearance therefore significantly exceeds -d[Ca2+]/dt. However, in this case the clearance rate obtained from a higher transient can be used as a good estimate. If the assumption of Eq.1 is satisfied, the clearance function is obtained as the envelope of the recorded return curves in the d[Ca2+]/dt vs. [Ca2+] plot. The calcium influx rate Jinflux(t) during each transient is then estimated by substracting Jclearance([Ca2+](t)) from the measured rate d[Ca2+]/dt. We tested the adequacy of this procedure using surrogate calcium dynamics data.To separate the two fluxes, we first estimate the clearance function cay rate . In contJclearance is not solely dependent on the cytoplasmic calcium concentration, as was assumed above. In this case, the method described above can still be applied to experiments performed in presence of Thapsigargin or cyclopiazonic acid (CPA), to avoid release or uptake of Ca2+ by the ER. Comparison of the estimated fluxes from the experiments with and without Thapsigargin/CPA can be used to investigate the ER-dependent calcium fluxes. We apply this method to freshly isolated magnocellular neurons, in which we used Fura-2AM to measure the cytoplasmic [Ca2+] during depolarization-evoked Ca2+ transients of various amplitudes and durations; depolarization was induced [+ concentration.For cells in which the endoplasmic reticulum (ER) noticeably contributes to the calcium transient , the cle induced  by chang2+ fluxes may be used also in other cell types to help characterize the contribution of individual mechanisms to calcium dynamics.Our method of estimating the Ca


Here we present evidence for the dynamic interaction between the SOCE-associated regulatory factor (SARAF), STIM1 and Orai1. SARAF overexpression attenuated SOCE and the STIM1-Orai1 interaction in cells endogenously expressing STIM1 and Orai1 while RNAi-mediated SARAF silencing induced opposite effects. SARAF impaired the association between Orai1 and the Orai1-activating small fragment of STIM1 co-expressed in the STIM1-deficient NG115-401L cells. Cell treatment with thapsigargin or physiological agonists results in direct association of SARAF with Orai1. STIM1-independent interaction of SARAF with Orai1 leads to activation of this channel. In cells endogenously expressing STIM1 and Orai1, Ca2+ store depletion leads to dissociation of SARAF with STIM1 approximately 30s after treatment with thapsigargin, which paralleled the increase in SARAF-Orai1 interaction, followed by reinteraction with STIM1 and dissociation from Orai1. Co-expression of SARAF and either Orai1 or various N-terminal deletion Orai1 mutants did not alter SARAF-Orai1 interaction; however, expression of C-terminal deletion Orai1 mutants or blockade of the C-terminus of Orai1 impair the interaction with SARAF. These observations suggest that SARAF exerts an initial positive role in the activation of SOCE followed by the facilitation of SCDI of Orai1.Ca This mechanism is initiated by the depletion of the intracellular Ca2+ stores, mainly the endoplasmic reticulum (ER). The two major components of SOCE are STIM112+ sensor of the ER that communicates the signal of store depletion to the plasma membrane resident Ca2+ channel, and the channel pore-forming Orai134Store operated Ca2+-selective, inward-rectifying, channel that has been reported to be regulated by Ca2+ in two possible ways named fast Ca2+-dependent inactivation (FCDI), which occurs within milliseconds62+-dependent inactivation (SCDI) that inactivates Orai1 over 2–3 min to prevent Ca2+ overload82+ entering through Orai1 channels, and constitutes a slow negative feedback process. SCDI has been reported to include both store refilling-dependent and independent components in Jurkat T cellsOrai1 is a highly Ca2+ dissociation, leads to oligomerization and clustering of STIM1 into puncta located at the ER–plasma membrane junctions2+ bound intraluminal region remains monomeric2+ store depletion, the N-terminal region of STIM1 releases Ca2+ and oligomerizes resulting in the conformational change that releases the SOAR domainSTIM1 N-terminus exhibits an EF-hand motif, which, upon Ca2+ store depletion2+, the CTID lobes facilitate access of SARAF to SOAR to keep SOAR in an inactive state, while store depletion results in an initial dissociation of SARAF, to allow activation of STIM1 and SOCE, followed by a reinteraction of SARAF with SOAR, facilitated by the STIM1(490–530) lobe, a process that has been associated with the SCDI of Orai1Recent studies have identified a C-terminal inhibitory domain  downstream of the SOAR domain, whose deletion leads to spontaneous clustering of STIM1 and activation of Orai1 in the absence of Ca2+ entry, and then reinteracts with STIM1 to mediate SCDI. These findings shed new light on the regulatory mechanism of SOCE.In the present study we have investigated the interaction of SARAF with STIM1 and Orai1 during the activation of SOCE. Our results indicate that SARAF is a modulator of the interaction between STIM1 and Orai1 channels that, during the initial steps of the activation of SOCE, transiently dissociates from STIM1 to associate with the C-terminus of Orai1 to activate Ca2+-free medium resulted in a transient rise in cytosolic Ca2+ concentration ([Ca2+]c) due to passive Ca2+ release from the intracellular stores. The subsequent SOCE, upon readdition of Ca2+ to the medium, was significantly inhibited by 45% in cells overexpressing SARAF (p < 0.001). As expected, SOCE was significantly enhanced by 20% in cells where endogenous SARAF levels were reduced by siRNA  and had a negligible effect on this interaction in resting cells. Analysis of the specificity of the anti-Orai1 and anti-STIM1 antibodies is depicted in the To further explore the mechanism underlying the regulation of SOCE by SARAF we modified the expression of SARAF in MEG-01 cells endogenously expressing STIM1 and Orai12+ store-depleted cells, we analyzed the interaction between Orai1 and the Orai1-activating small fragment  of STIM1 in NG115-401L cells, which express a negligible amount of endogenous STIM1202+ entry independently of Ca2+ store depletion as compared to mock-treated cells, which did not display constitutive Ca2+ entry. Ca2+ entry induced by co-expression of Orai1 and OASF was impaired by overexpression of SARAF and enhanced by SARAF silencing . Resting MEG-01 cells or cells stimulated with TG (1 μM) for 30 or 120 s were subjected to PLA. As shown in It has been suggested that SARAF is associated with STIM1 in resting cells and upon store depletion undergoes dissociation of STIM1 followed by reinteraction to activate SCDIWe further explored the interaction between SARAF and both STIM1 and Orai1 by co-immunoprecipitation. As shown in In order to explore whether the interaction SARAF-Orai1 requires STIM1 we repeated the study in NG115-401L cells lacking STIM1. As reported in 2+ entry in NG115-401L cells transfected with expression plasmid for Orai1 in combination with SARAF overexpression or siSARAF. Since NG115-401L cells release Ca2+ in response to TG but do not display SOCE2+ entry. As depicted in 2+-free medium resulted in a transient rise in [Ca2+]c due to Ca2+ release from the intracellular stores. Overexpression of Orai1 alone or in combination with either SARAF silencing or overexpression did not significantly modify the ability of ATP to release Ca2+ from the stores. If we consider the entry of Ca2+ stimulated by ATP, Orai1 overexpression resulted in a significant increase in Ca2+ influx to 187 ± 12% of control . As depicted in 2+ into the stores but SARAF silencing significantly enhanced the amount of Ca2+ released using ionomycin by 20% (p < 0.05) as previously described in HeLa and Jurkat cellsTo understand the functional significance of the interaction of SARAF with Orai1 independently of STIM1 we determined Ca1–38, ΔN1–72 and ΔN1–89) or the C-terminal deletion mutant  together with SARAF. As shown in Finally, we have investigated the location of the possible SARAF binding site of Orai1 in NG115-401L cells overexpressing pEYFP-Orai1 (full-length), the N-terminal deletion mutants  as compared to the introduction of an antibody that recognizes 18 amino acids located in the N-terminal region . The presence of these antibodies inside the cells was investigated by immunoprecipitation without adding any additional anti-Orai1 antibody and subsequent Western blotting with the anti-Orai1 antibody 2+ store discharge probably to interact with Orai1 and cooperate with STIM1 in the activation of Orai1 and, then SARAF rapidly re-associates with STIM1 (and dissociates from Orai1) to participate in the inactivation of SOCE. Our results indicate that SARAF interacts and activates Orai1 channels in a scenario that allows direct association of both proteins independently of STIM1, which has been reported to be negative regulated by SARAF17The present study provides relevant information concerning the mechanism of SOCE regulation by SARAF. We have found for the first time that SARAF associates with Orai1 and enhances Orai1 function in STIM1-deficient cells. SARAF interacts with the C-terminal region of Orai1 since Orai1 constructs lacking the C-terminus are unable to associate with SARAF and blockade of this region also impairs its ability to interact with SARAF. Our results suggest that SARAF dissociates from STIM1 upon Ca2+ and leukotriene C4-regulated Ca2+ channels23242+ (SOC) channels, also involving Orai1 subunits22262728292+ store depletion requires dissociation of SARAF to allow SOAR to interact with and activate Orai1 channels. Subsequently, SARAF associates again with STIM1 to mediate SCDI. These findings are based on the observation that Orai1 current activated by the constitutively active STIM1(4E/4A) and STIM1(D76A) mutants was unaffected by SARAF, as well as on the role of different STIM1 mutants on SCDI of Orai1 currents2+ store depletion reaching a minimum after 30s of stimulation and then reinteracts with STIM1, and opposite results were observed when we tested the interaction of SARAF with Orai1, revealing that 30s after the initiation of Ca2+ store depletion there is an increase in SARAF-Orai1 co-immunoprecipitation followed by a decrease in the association between both proteins. Using STIM1-deficient NG115-401L cells we have found that dissociation of SARAF and Orai1 observed 30 s after treatment with TG occurs independently of STIM1. The dynamic interactions of SARAF with STIM1 or Orai1 were found to occur with the same time courses, which might suggest that SARAF dissociates from STIM1 in the initial stages of Ca2+ store depletion to interact with Orai1, although the mechanism underlying this translocation is unclear at this stage.Current evidence indicates that the Orai1 forming channels , rabbit polyclonal anti-β-actin antibody , and bovine serum albumin (BSA) were from Sigma . Rabbit polyclonal anti-TMEM66 (SARAF) antibody  and Turbofect transfection reagent were from Thermo Fisher . Mouse monoclonal anti STIM1 antibody  was from BD Transduction Laboratories . Calmidazolium chloride was from Bio-Rad . Rabbit polyclonal anti-Orai1 antibody , horseradish peroxidase-conjugated anti-mouse IgG antibody and anti-rabbit IgG antibody for IP (not recognizing the heavy and light chains of the immunoprecipitating antibody) were from Abcam . Rabbit polyclonal anti-Orai1 antibody  was from Prosci . Amaxa nucleofector kit C was purchased from . Protein A-agarose was from Upstate Biotechnology Inc. . Mouse anti-GFP antibody . All other reagents were of analytical grade.Fura-2 acetoxymethyl ester (fura-2/AM), was from Molecular Probes . Thapsigargin (TG), ATP, rabbit polyclonal anti-Orai1 antibody , DuolinkPlasmids were based on the previously published SARAF sequences (GenBank: JQ348891.1). The DNA of the complete cds was isolated from NG115-401L cells using specific primers . Final cDNA was purified and cloned into the EcoRV site previously inserted in the pIRES2-eGFP-RV expression vector. Nucleotide sequence of this construct was verified by sequencing.To knockdown expression of SARAF1, a pLKO.1-puro plasmid–based shRNA targeting the sequence: CGGACTTAGATATTGCATACA  was used (SARAF-shRNA). In addition, a non-targeting shRNA plasmid (NT-shRNA) that targets no known human sequence was used as a control. A primer containing the target sequence along with a stem loop followed by the reverse target sequence was annealed to a complimentary primer and inserted into the EcoRI and AgeI sites of the pLKO.1-puro plasmid . The resulting hairpin consisted of the following sequence: 5′-CCGGCGGACTTAGATATTGCATACACTCGAGTGTATGCAATATCTAAGTCCGTTTTTTG-3′. The correct insertion of the hairpin into pLKO.1 plasmid was finally checked by sequencing.2 in RPMI or DMEM, respectively, supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine and 100 U/mL penicillin and streptomycin.The megakaryoblastic cell line MEG01 was used in this study as a cellular model endogenously expressing STIM1. Moreover, STIM1-deficient NG115-401L cells were used for certain experimental procedures to avoid interference with endogenous STIM1. MEG01 and NG115-401L cell lines were obtained from ATCC  and Sigma , respectively, and cultured at 37 °C with a 5% CO1–38, ΔN1–72 and ΔN1–89) or the pEYFP-Orai1 C-terminal deletion mutant  kindly provided by Dr. Romanin, as well as with the siSARAF or scramble plasmid as described previously3637Cells were transfected with expression plasmids for pIRES2-EGFP-SARAF, pEYFP-Orai1, pEYPF-OASF, the pEYFP-Orai1 N-terminal deletion mutants (Orai1 ΔNCell transfection with shOrai1 was performed as described previously340/F380) was calculated pixel by pixel and data are presented as ΔF340/F380. ATP-evoked Ca2+ influx was measured as the integral of the rise in [Ca2+]c above basal for 1½ min after the addition of ATP in the presence of external Ca2+, corrected by subtraction of the integral over the same period for stimulation in the absence of external Ca2+ (with 100 μM EGTA).Cells were loaded with fura-2 by incubation with 2 μM fura 2/AM for 30 min at room temperature. Coverslips with cultured cells were mounted on a perfusion chamber and placed on the stage of an epifluorescence inverted microscope  with image acquisition and analysis system for videomicroscopy . Cells were continuously superfused with HEPES-buffered saline (HBS) containing (in mM): 125 NaCl, 5 KCl, 1 MgCl2, 5 glucose, 25 HEPES, and pH 7.3, supplemented with 0.1% (w/v) BSA.. Cells were alternatively excited with light from a xenon lamp passed through a high-speed monochromator  at 340/380 nm. Fluorescence emission at 505 nm was detected using a cooled digital CCD camera  and recorded using Aquacosmos 2.5 software . Fluorescence ratio (F6 cell/mL) were lysed with an equal volume of ice-cold 2×NP-40 buffer, pH 8, containing 274 mM NaCl, 40 mM Tris, 4 mM EDTA, 20% glycerol, 2% nonidet P-40, 2 mM Na3VO4 and complete EDTA-free protease inhibitor tablets. Aliquots of cell lysates (1 mL) were immunoprecipitated by incubation with 1 μg of anti-STIM1 or 2 μg of anti-SARAF or anti-Orai1 antibody and 25 μL of protein A-agarose overnight at 4 °C on a rocking platform. The immunoprecipitates were resolved by 8% SDS-PAGE and separated proteins were electrophoretically transferred onto nitrocellulose membranes for subsequent probing. Blots were incubated overnight with 10% (w/v) BSA in tris-buffered saline with 0.1% Tween 20 (TBST) to block residual protein binding sites. Immunodetection of Orai1, STIM1 and SARAF was achieved by incubation for 2h with anti-STIM1 antibody diluted 1:250 in TBST, anti-Orai1 antibody diluted 1:200 in TBST, the anti-SARAF antibody diluted 1:1000 in TBST and anti-calmodulin antibody diluted 1:500. The primary antibody was removed and blots were washed six times for 5 min each with TBST. To detect the primary antibody, blots were incubated for 1 h with horseradish peroxidase-conjugated goat anti-mouse IgG antibody or horseradish peroxidase-conjugated goat anti-rabbit IgG antibody diluted 1:10000 in TBST and then exposed to enhanced chemiluminiscence reagents for 4 min. The density of bands was measured using C-DiGit Chemiluminescent Western Blot Scanner. Data were normalized to the amount of protein recovered by the antibody used for the immunoprecipitation.The immunoprecipitation and Western blotting were performed as described previously®In Situ Red Starter Kit Mouse/Rabbit and performed following the manufacturer’s instructions and as previously described®In Situ PLA Probe anti-rabbit PLUS or Duolink®In Situ PLA Probe anti-mouse MINUS) conjugated to unique oligonucleotides. The cultures were further subjected to in situ PLA using a Duolink Detection kit according to the manufacturer’s instructions. Briefly, the slides were incubated with Ligation-Ligase solution in a pre-heated humidified chamber for 30 min at 37 °C, followed by incubation with amplification polymerase solution for an additional 100 min at 37 °C. Finally, the cells were washed in Wash Buffer B, and the slides were mounted using Mounting Medium with DAPI® and evaluated using a Eclipse TE300 fluorescence microscope with a ×60 oil-immersion objective . The quantification of PLA signals was performed using ImageJ software . The processed images were thresholded and the in situ PLA signals per cell were quantified. The settings were kept constant for all of the images throughout the experiments. Quantifications were performed from 30 to 40 images from a minimum of four slides for each treatment corresponding to 100–200 cells. Mouse anti-Orai1 directed towards the C-terminus  combined with rabbit anti-Orai1 directed towards 18 amino acids located in the N-terminal region of human Orai1  was used as a positive control. As a negative technical control, the primary antibodies were used alone, or both primary antibodies were omitted. The negative controls did not yield any significant PLA signals at any treatment condition.The PLA was performed using the Duolinkt test was used. P < 0.05 was considered to be significant for a difference.Analysis of statistical significance was performed using one-way analysis of variance. For comparison between two groups Student’s How to cite this article: Albarran, L. et al. Dynamic interaction of SARAF with STIM1 and Orai1 to modulate store-operated calcium entry. Sci. Rep. 6, 24452; doi: 10.1038/srep24452 (2016).


TRH-induced membrane depolarization was reduced in the presence of intracellular BAPTA, also in media containing nominally zero [Ca2+]o, suggesting a critical role for both intracellular Ca2+ release and Ca2+ influx. TRH-induced inward current was unchanged by T-type Ca2+ channel blockade, but was decreased by blockade of high-voltage-activated Ca2+ channels (HVACCs). Both the pharmacologically isolated GIRK-like and the TRPC-like components of the TRH-induced response were decreased by nifedipine and increased by BayK8644, implying Ca2+ influx via L-type Ca2+ channels. Only the TRPC-like conductance was reduced by either thapsigargin or dantrolene, suggesting a role for ryanodine receptors and Ca2+-induced Ca2+ release in this component of the TRH-induced response. In pituitary and other cell lines, TRH stimulates MAPK. In PVT neurons, only the GIRK-like component of the TRH-induced current was selectively decreased in the presence of PD98059, a MAPK inhibitor. Collectively, the data imply that TRH-induced depolarization and inward current in PVT neurons involve both a dependency on extracellular Ca2+ influx via opening of L-type Ca2+ channels, a sensitivity of a TRPC-like component to intracellular Ca2+ release via ryanodine channels, and a modulation by MAPK of a GIRK-like conductance component.In rat paraventricular thalamic nucleus (PVT) neurons, activation of thyrotropin-releasing hormone (TRH) receptors enhances neuronal excitability via concurrent decrease in a G protein-coupled inwardly rectifying K (GIRK)-like conductance and opening of a cannabinoid receptor-sensitive transient receptor potential canonical (TRPC)-like conductance. Here, we investigated the calcium (Ca Also, isponsive . In lateng media . By contar BAPTA . Here, iar BAPTA , we soug2+ currents (IT), hyperpolarization-activated cation currents (Ih), and undefined potassium conductances (2-5% CO2) and cooled (<4°C) in artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 127 NaCl, 3.1 KCl, 1.3 MgCl2, 2.4 CaCl2, 26 NaHCO3, and 10 glucose . Brain slices 350–400 μm in thickness were cut in the coronal plane with a Vibratome 3000 Plus Sectioning System , incubated in gased ACSF for >1 h at room temperature, and then transferred to a recording chamber and superfused (2–4 ml/min) with oxygenated ACSF at 32–34°C. In all experiments, the blind-patch technique was used for whole cell current-clamp and voltage-clamp recordings using either an Axopatch 1D or Axopatch 200A, respectively . Data were filtered at 2 kHz, continuously monitored and stored on disk. A Digidata 1200B interface with Clampex software  was used to generate current and voltage commands, as well as to store data. Recordings were obtained from PVT neurons with borosilicate thin-walled micropipettes (resistances of 3–7 MΩ) filled with (in mM) 130 K gluconate, 10 KCl, 2 MgCl2, 10 HEPES, 1 EGTA, 2 Mg-ATP, 0.3 Na-GTP . Pipettes had resistances of 4–7 MΩ. Access resistance <15 MΩ was considered acceptable. To eliminate the contribution of Na+-dependent conductances, we used ACSF where NaCl was replaced with Tris-Cl (2+) was utilized to eliminate the contribution of GIRK channels to TRH-induced inward currents . Voltage-clamp analyses used a standard ramp protocol (step from −63 mV to −43 mV for 0.5 s and then ramped from −43 mV to −113 mV or −123 mV in 3 s followed by step back to −63 mV) from a holding potential of −63 mV. The net TRH-induced current was obtained by subtracting the control ramp response from that obtained during the TRH-induced response. Off-line analyses were performed using Clampfit version 9 or 10 . As recently reported, TRH-induced inward currents in PVT neurons rapidly desensitize (P < 0.05 and better) were determined using both the paired or unpaired Student's t-test and ANOVA .Experimental protocols were approved by the Ottawa Hospital Research Institute Animal Care and Use Committee. We used 3–8-wk-old Wistar rats of either sex that were housed in pairs in a temperature-controlled (22–24°C) environment with a 12:12-h light-dark (LD) cycle, with food and water available ad libitum. Our earlier studies of anterior PVT neurons revealed a diurnal variation in some of their intrinsic properties, notably resting membrane potential, low threshold-activated Cauctances . To mini Tris-Cl . ACSF cocurrents . Series currents . Membranensitize . TherefoPD98059 were purchased from Sigma Chemical . Tetrodotoxin (TTX) was purchased from Alomone Labs . Z941 was kindly provided by Dr. Terry Snutch from University of British Columbia, Canada. Most drugs were bath applied at the concentrations indicated.BayK8644, ω-Agatoxin IVA, thapsigargin, dantrolene, and W7 were purchased from Tocris Bioscience . Nifedipine, BAPTA, ω-conotoxin GVIA, and A, B, D; n = 5; one-way ANOVA, F = 17.024, P < 0.001). There was no difference in resting membrane potential between these two groups . To evaluate a dependency on Ca2+ influx, we next used ACSF with nominally zero Ca2+. Here, the TRH-induced depolarization was also significantly reduced . The data suggest major roles both for Ca2+ influx and intracellular Ca2+ in the TRH-induced response.We first evaluated the TRH response under current-clamp conditions. During intracellular dialysis with 10 mM BAPTA in the pipette media, the membrane depolarization induced by a brief bath application of TRH (100 nM for 60 s) was significantly reduced when compared with controls  was significantly reduced . Because TRH responses occurred from a holding potential around −63 mV, a voltage range at which low-voltage activated T-type Ca2+ channels are fully functional . However, the TRH-induced current was significantly reduced during bath application of cadmium , a nonselective blocker of high-voltage activated Ca2+ channels (HVACCs) . While Cd2+ at this concentration was reported to impair T-type Ca2+ function in cultured thalamic neurons , a potential contribution from N-, P-, and Q-type channels would seem unlikely.Recordings in voltage-clamp mode in the presence of 1 μM TTX confirmed a dependency of the TRH-induced inward current on external Caonal see , we nextchannels . Whereas current , the TRH neurons , our obsrons see . On the 2+ channels can be activated at low hyperpolarized voltages . Conversely, in the presence of BayK8644 (10 μM), an activator of L-type Ca2+ channels  and prolonged in duration  and an activator (BayK8644) under recording conditions that blocked either the contribution of TRPC-like channels (low Na+) or GIRK-like channels (1.3 mM Ba2+) to the TRH-induced response . Under these experimental conditions, not only was the TRH-induced current significantly decreased , but the net TRH current now reversed at −105 mV, close to the K+ equilibrium potential . However, when tested in ACSF containing 1.3 mM Ba2+ , suggesting an involvement of CICR in TRH-induced opening of TRPC-like channels.The observation that chelation of intracellular Ca of CICR . Therefootential A. This siminated , the res3Rs) or ryanodine receptors (RyRs). Data from our previous investigation indicated that IP3Rs are unlikely to be involved in the TRH-induced inward current . In addition, the TRH response was unchanged in low Na+ ACSF  but significantly smaller in ACSF containing 1.3 mM Ba2+ . These data suggest that CICR through functional coupling between L-type Ca2+ channels and RyRs contributes to the TRH-induced activation of TRPC-like channels.CICR can be mediated through activation of either inositol 1, 4, 5-trisphosphate receptors , suggesting that activation of MAPK could be involved in the regulation of TRH-induced decrease of a GIRK-like conductance. In support of this, we noted that the TRH-induced current was significantly reduced when PD98059 was applied under conditions in which the activity of TRPC-like channels was inhibited . By contrast, when tested in ACSF containing 1.3 mM Ba2+, PD98059 did not alter the TRH-induced increase in TRPC-like channel conductance , suggesting that MAPK only participates in the regulation of GIRK channels.Investigations in various endocrine-like cell lines reveal that activation of TRH receptors can lead to stimulation of MAPK , 41. To 2+]i), with a first phase of the response involving Ca2+ release from intracellular stores, followed by a delayed prolonged elevation of [Ca2+]i through influx via voltage-gated Ca2+ channels . While Cd2+ at the concentration used here has been reported to impair T-type channel current in cultured thalamic neurons . 2-AG is perhaps the most abundant endocannabinoid in the rat brain 77745. L. P. Renaud held the GlaxoSmithKline/CIHR/J. David Grimes Chair at the University of Ottawa.This research was supported by the No conflicts of interest, financial or otherwise, are declared by the authors.M.K., L.Z., and L.P.R. conception and design of research; M.K. and L.Z. analyzed data; M.K., L.Z., and L.P.R. interpreted results of experiments; M.K. and L.Z. prepared figures; M.K. and L.P.R. drafted manuscript; M.K. and L.P.R. edited and revised manuscript; M.K., L.Z., and L.P.R. approved final version of manuscript; L.Z. performed experiments.