Effect Of Orai1 On Lipopolysaccharide-related Calcium Overload

BackgroundOne core event to all of the gram-negative bacterial sepsis complications including disseminated intravascular coagulation (DIC), vascular scleratheroma, systemic vascular collapse, multiorgan dysfunctional syndrome (MODS), acute respiratory disgtress syndrome (ARDS) is endothelial cell (EC) injury and dysfunction. The vascular endothelium serves as the key barrier between the intravascular compartment and extravascular tissues and plays a critical role in a large number of physiological and pahtological processes. ECs, integrally involved in regulating blood flow, coagulation, leukocyte trafficking, edema formation, wound healing, and angiogenesis, are constantly exposed to circulating mediators such as bacterial lipopolysaccharide (LPS) or endotoxin, a highly proinflammatory molecule that is a component of the outer envelope of all gram-negative bacteria. Evidence exists that LPS alone or in concert with other endogenous factors, is responsible for much of the EC injury and dysfunction associated with gram-negative sepsis. LPS-induced EC programmed cell death or apoptosis is an event that is believed to contribute to the pathogenesis of sepsis and its complications.More recent studies have shown that alterations in the intracellular Ca2+ homeostasis is commonly involved in the initiation of apoptosis. Oscillation of intracellular Ca2+, intracellular Ca2+ linking stores such as the endoplasmic reticulum (ER) and/or mitochondria linked to the cytoplasmic membrane or intracellular Ca2+ overload, can cause cytotoxicity and trigger apoptotic cell death, but the mechanism is unclear.Several studies have also revealed that store-operated Ca2+ (SOC) influx is the major mechanism for Ca2+ entry in many non-excitable cell types. SOC entry is the process whereby modest ER Ca2+ store depletion leads to activation of the plasma membrane (PM) Ca2+ release-activated channel (CRAC) activation providing a sustained Ca2+ elevation in the cytoplasm from the extracellular matrix. Despite more than a few decades of research, little is known about the mechanism of the channels responsible for SOC/CRAC entry. Recently, membrane proteins, stromal-interacting molecule 1 (STIM1) and Ca2+ release-activated Ca2+ modulator 1 (Orai1/CRACM1), were each shown to be essential for the activation of SOC/CRAC channels. STIM1 is likely the Ca2+ "sensor" of ER Ca2+ stores, moving in response to restore Ca2+ depletion into ER puncta close to the plasma membrane (PM). Orai1/CRACM1 was identified in severe combined immunodeficiency (SCID) as an essential subunit for activating CRAC channels.Thus, Orail and STIM1 together function to reconstitute store-operated calcium channels.Several studies revealed that Btk/Tec kinases, a family of non-selective receptor tyrosine kinases that include Tec, Btk, Itk, Bmx, and Txk, specifically regulate the TCR or BCR-dependent sustained phase of calcium signaling. Btk-deficient B-lymphocytes from humans with X-linked agammaglobulinemia (XLA) and mice with X-linked immunodeficiency (XID) exhibit diminished inositol 1,4,5-trisphosphate production and calcium mobilization. Btk/Tec kinases mediating sustained calcium signaling are required for phosphorylation of PLCyl. Studies in mice deficient in Bruton's tyrosine kinase previously showed that Btk is phosphorylated following stimulation of human monocyte cell lines and primary human monocytes with LPS by directly binding TLR4 (Toll-like receptor 4) and/or inducing NF-κB activation. Previous studies have also suggested that the PLCy protein is required for the activation of store-operated Ca2+ channels as a molecular component of agonist-initiated Ca2+ entry mechanism, independent of its role in the generation of IP3. However, PLCyl dose not interact with STIM1 in H4â…¡E cells.Here we show that CRACM1/Orai1, a subunit of the SOC/CRAC channel, and STIM1, the "sensor" of Ca2+ with ER Ca2+ stores, were induced by LPS in a dose-and time-dependent manner. The elevation of intracellular Ca2+ levels in HUVECs, and Ca2+ overload and cell apoptosis induced by LPS were attenuated by Orail and STIM1 siRNA-mediated knockdown, and also in part inhibited by Btk and/or PLCy knockdown. We also showed that STIM1 interacted with phospho-PLCy, a key molecule downstream of Tec kinase signaling that mediates sustained calcium influx induced by LPS. LFM-A13 (a Btk inhibitor),2-APB (a SOC/CRAC inhibitor) or U73122 (a PLC inhibitor) at least in part abrogated the Ca2+ overload and cell apoptosis induced by LPS. Furthermore, the results of immunoprecipiation suggested LPS likely facilitates the association of SOCE with ROCE by the link of STIM1 to IP3R. Together, these data support a model whereby the SOC/CRAC channel is required for LPS-induced Ca2+ overload and cell apoptosis and is possibly associated with Btk/Tec and PLCy-mediated ROCE calcium signals.Methods1. Cellular apoptosis and immunhistochemistry. HUVECs (3×106) growing in 6-well plate were treated with LPS (0,0.01,0.1,1.0 or 10μg/ml), and DNA fragmentation was assessed at the indicated times of treatment. DNA fragmentation due to internucleosomal cleavage was determined as described previously. HUVECs (1×106) were cultured on coverslips in the 6-well plates for 24 h to allow cells to grow to 50-60% confluency with the addition of fresh media. The HUVECs were then treated with LPS for various time points and fixed with 95% ethanol, 5% glacial acetic for 3-5 min and rinsed thoroughly in PBS each time. Endogenous peroxidase was blocked by incubating in 3% H2O2 in PBS for 30 min and then blocked with 2-5% normal serum in PBS for 1 h. After incubation with anti-Caspase-3 (Santa Cruz, CA), routine immunocytochemistry procedures ans analysis were performed as described previously. Cells exhibiting the brown cytoplasmic staining from the colorimetric reaction were considered positive for active Caspase-3. Negative controls, lacking the labeling enzyme, yielded no reaction product. Caspase-3 staining intensity for image quantification was measured as described previously. For slides that were used for image quantification, no counterstain was applied in order to simplify image colorimetric quantitation.2. siRNA design and construction and real-time RT-PCR. The 21-nucleotide duplexed STIM1, CRACM1, Btk/Tec and PLC siRNAs were designed by GenScript Corporation, and the specificity checked by BLAST searches against the NCBI (National Center for Biotechnology Information) database. The RNA sense sequences of the human transcripts were:STIM1,5'-GAAGCTGCGTGACGAGATC-3'(NM₀03156); PLC-γ1, 5'-CCGAGAGGATCGTATATCA-3'(GenBank accession no. NM₀13187); Btk, 5'-GAGTAACATTCTAGATGTGATGG-3'(GenBank accession no. NM₀00061); and CRACM1,5'-TATGGCTAACCAGTGAGCGGT-3'(NM₀32790). The negative or non-silencing control siRNA had no significant sequence homology to any known human gene. The synthesized sense and antisense siRNAs oligos were resuspended annealed inserted into the pRNA-CMV3.1/Hygro vector according to the manufacturer's instructions. Human PLC-γsiRNA plasimids were purchased from Santa Cruz. Gene-specific mRNAs were quantitated by reverse transcription of 2μg of total cellular RNA using random primers, followed by real-time PCR with gene-specific primers and probes which were designed based on the cDNA sequences of the target genes. The products of each PCR cycle were quantitated using the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA USA) and normalized against endogenous 18s ribosomal RNA using TaqMan Ribosomal RNA Control Reagents (Applied Biosystems). Results were analyzed and shown as fold changes (arbitrary unit) relative to a control group.3. Measurements of [Ca2+]i using fluo-3/AM. The HUVECs (0.1×106) cells were treated with LPS at the indicated concentrations followed by measurement of intracellular free calcium ([Ca2+]i). Fluo-3/AM (10μM) (Biotium, Calif, USA) was added into the medium and incubated in the dark for 50 min at 37℃. Cells were washed free of extracelluar Fluo-3/AM dye in standarded external solution containing (in mM) NaCl 145, KCl 2.8, CaCl2 2, MgCl2 2, D-glucose 10, and HEPES 10, and adjusted to pH7.4 with NaOH. Ca2+-free solution was a standard external solution in which the Ca2+ was substituted with 1 mM EGTA. Fluorescence measurements of [Ca2+]i were performed using confocal laser scanning microscopy (Olympus FV500, Japan) with an Olympusâ…¨71 camera in the pressence or absence of Ca2+ in the bath. Fluo-3/AM was excited at 488nm and emission measured between 500 and 550 nm. Fluorescence intensity and the curve of the time course were analyzed automatically by the computer software. Increases in [Ca2+]i is expressed as a ratio:fluorescence intensity of Fluo-3 over baseline (fluorescenece/baseline). The peak [Ca2+]i over baseline was also measured.4. Immunnoprecipitation of non-phosphorylated and phosphorylated proteins and western blot analysis. Cells (1×107) were lysed for 20 min on ice in a lysis buffer (pH 8.0) containing 200 mM boric acid,5 mM NaF, 0.5% Triton X-100, 5 mM EDTA,150 mM NaCl, and protease inhibitors of leupeptin (10μg/ml) and aprotinin (10μg/ml) and phenylmethylsulfonyl fluoride (1 mM). Cell lysates were centrifuged at 2500 rpm at 4℃for 15 min, and supernatants were used as whole cell lysates for Western blotting and immunoprecipitation. For Western blots, protein samples were resolved by SDS/PAGE and transferred onto nitrocellulose membranes. The membranes were then incubated for 1-2 h at room temperature in blocking buffer [TBST(Tris-buffer saline and 0.1% Tween-20) containing 5%(w/v) non-fat milk powder]. The membranes were then probed with the following primary antibodies:rabbit anti-Orai1 antibody (1:200); rabbit anti-STIM1 antibody (1:400); rabbit anti-PLC-γ1 antibody (1:400); rabbit anti-Btk/Tec antibody (1:500); anti-p-Btk (1:800) and anti-IP3R (1:400) (Santa Cruz Biotechnology) in blocking Buffer at room temperature for 2h. The membranes were then washed three times with TBST, followed by incubation of the membranes with secondary antibody (goat-anti-rabbit IgG-HRP-conjugate diluted 1:10000 in blocking buffer) at room temperature for 1 h. For immunoprecipitation, 200μg of whole cellular lysates were incubated with 5μg/ml anti-Btk (Q20; Santa Cruz Biotechnology) for Btk/Tec or 5μg/ml anti-PLCγ(Q20; Santa Cruz Biotechnology) for PLCy (5μg/200μg of cell lysates) followed by incubation with protein A/G plus agarose overnight at 4℃. All samples were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Phosphorylated immunoprecipitates were assessed by Western blotting with the 4G10 anti-phosphotyrosine antibody (anti-p-Tyr). Western blotting analysis was performed using standard procedures as previously described. ECL was used for antibody detection according to the manufacturer's instructions (Santa Cruz Biotechnology).5. Flow cytometric analysis Cells undergoing apoptosis were detected by double staining with Annexin V-FITC/PI in the dark according to the manufacturer's instructions. Cells attached to dishes were harvested with 0.25% trypsin and washed twice with cold PBS. Cell pellets were suspended in 1×binding buffer (10 mM HEPES/NaOH, pH 7.4,140 mM NaCl,2.5 mM CaCl2) at a concentration of 1×106 cells/ml. Then the cells were incubated with Annexin V-FITC and propidium iodide (PI) for 15 min (22-25℃) in the dark. The stained cells were immediately analyzed by flow cytometry (FAS Calibur BD, USA). Each measurement was carried out in at least triplicate.6. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay To characterize inhibitory effect of 2-APB and U73122 against LPS-induced cell death, we examined TUNEL assay and activation of caspase 3. An in situ Apoptosis Detection Kit (Roche, Germany) was used to carry out TUNEL staining on 4-μm-thick paraffin sections according to the manufacturer's instructions. Forty fields from each section at light microscopy were randomly selected under high power views (×400), and the number of TUNEL-positive cells or was calculated.7. The animal model and pathological evaluation.Sixty C57BL/6 mice, eight to ten weeks old, weight 16-25g. Adaptive feeding 7-10 days, standard mice food, free for the water. Take the first 36 were separated randomly into LPS+2-APB group, LPS+U73122group, mice in each group were killed and obtained lung respectively at Oh, 1h,6h,12h,24h,48h after operation. The remaining 24 were randomly divided into control group, LPS group, LPS+2-APB group, LPS+U73122 group, mice in each group were killed and obtained lung respectively at 12h after operation. Mice were anesthetized with isoflurane (1.5% isoflurane concentration, oxygen flow 2L/min).3mg/kg by nasal inhalation of 2mg/ml of LPS (control group, normal saline inhalation), then put mice at the vertical position for 1 minute to prevent the liquid back to the airways. Mice in LPS+2-APB group were injected intraperitoneally at 2mg/kg dose of 2-APB (dissolved in DMSO). Mice in LPS+U73122 group were injected intraperitoneally at 30mg/kg dose of U73122 (dissolved in DMSO). The remaining mice were not injected. The mice back into the cage, free for the water. Lung specimens were examined for pathological and evaluated by Szapiel's double-blind pathological score.Results1. Intracellular calcium homeostasis dysfunction and cellular apoptosis induced by LPS in HUVECs.The results of calcium imaging/fluorescence intensity showed that LPS induces intracellular[Ca2+]i increase in HUVECs in a dose-dependent manner,but peaked at 24h in "1.0μg/ml" group. The results of immunohistochemical staining for active Caspase-3 as well as detection of low-molecular-weight DNA fragmentation indicated that LPS induced cell apoptosis in a time-/dose-dependent manner and "1.0μg/ml" group with [Ca2+]i overload had a higher cell apoptosis ratio than that of control. These results suggested that LPS-stimulated increase in intracellular Ca2+ increase and disturbance of cellular homeostasis likely triggers the apoptotic process.2. SOC/CRAC channels orchestrate [Ca2+]i increase and cell apoptosis induced by LPS in HUVECs.The results of real-time RT-PCR and Western blot showed that Orai/CRACM1 and STIM1 express in endothelial SOCE. SiRNAs against either STIM1 or Orail markably attenuate thapsigargin-triggered SOC Ca2+ entry in VECs. SOCE was greatly inhibited after transfecting cells with STIM1 or Orail siRNAs compared to cells transfected with control siRNAs. The intracellular Ca2+ entry curve showed that [Ca2+]i induced by LPS was remarkably inhibited by siRNAs against Orail or STIM1 as well as co-transfection with both siRNAs. The results in ELISA and IHC show that induction of cell apoptosis by LPS was apparently reduced by siOrail or siSTIM1, and especially by siOrail and siSTIM1 co-transfection.These results indicated that CRACM1/Orai1, a subunit of the Ca2+ channel, and STIM1, the "sensor" of ER Ca2+ stores, were induced by LPS in a dose-and/or time-dependent manner. Furthermore, the elevation of intracellular Ca2+ levels in HUVECs, and Ca2+ overload and cell apoptosis induced by LPS were attenuated by co-transfection with Orai1 and STIM1 siRNAs.To further validate the effect of Orai1 on extracellular calcium influx and cell apoptosis, we used pcDNA3.0 vector mediated overexpression of Orai1, then transfected HUVEC cells, the transfection efficiency was verified by Western blot. Then we detected intracellular calcium influx. We examined TUNEL assay to characterize inhibitory effect of 2-APB and U73122. Results further validateed Orai1/CRACM1 as the core components in SOC calcium channel involving in LPS induced calcium overload and apoptosis.3. [Ca2+]i increase and cellular apoptosis are related to LPS-induced Btk and PLCy activation.The results of phosphorylated immunoprecipitation and RT-PCR together with the observation of a transient increase in [Ca2+]i induced by LPS showed that LPS induction and phosphorylation of Btk and PLCγcontribute to intracellular [Ca2+]i influx.4. Btk and PLCγ-mediated ROCE regulates intracellular [Ca2+]i overload and apoptosis induced by LPS.The results of RNAi and IHC and DNA fragmentation assay indicated that [Ca2+]i overload and apoptosis induced by LPS was reduced by with siBtk, siPLCγ1.5. SOCE and Btk with PLCγtogether mediate [Ca2+]i influx induced by LPS.The results of IP and [Ca2+]i and block assay of SCOE and ROCE indicated that SOC/CRAC channels are associated with PLCγ-mediated ROCE Ca2+ signals possibly via STIM1.6.2-APB and U73122 were used in vivo to validate the above results in vitro. We found lung injury and apoptosis were improved in LPS+2-APB group and LPS+U73122 group (compared with LPS group).2-APB (2mg/kg), U73122 (30mg/kg) could reduce LPS-induced apoptosis. These results suggest that Orail-mediated SOCE and PLCγ-mediated ROCE participated in the LPS-induced cell calcium overload and apoptosis.Conclusion1. Lipopolysaccharides (LPS) or endotoxins are responsible for intracellular calcium homeostasis dysfunction and cellular apoptosis in HUVECs.2. Orai/CRACM1 and STIM1 express in endothelial SOCE, and the elevation of intracellular Ca2+ levels in HUVECs, and Ca2+ overload and apoptosis induced by LPS are required for SOCE.3. Btk and PLCγ-mediated ROCE regulates intracellular [Ca2+]i overload and apoptosis induced by LPS.4. SOCE and ROCE together mediate [Ca2+]i influx induced by LPS.