ORMDL3 modulates store-operated calcium entry and lymphocyte activation

The asthma-associated ORMDL3 gene productregulates endoplasmic reticulum-mediatedcalcium signaling and cellular stress

Gerard Cantero-Recasens{, Cesar Fandos{, Fanny Rubio-Moscardo, Miguel A. Valverde

and Ruben Vicente�

Laboratory of Molecular Physiology and Channelopathies, Department of Experimental and Health Sciences,

Universitat Pompeu Fabra, Edifici PRBB, C/ Dr Aiguader 88, Barcelona 08003, Spain

Received September 9, 2009; Revised and Accepted October 7, 2009

Alterations of protein folding or Ca21 levels within the endoplasmic reticulum (ER) result in the unfolded-proteinresponse (UPR), a process considered as an endogenous inducer of inflammation. Thereby, understanding howgenetic factors modify UPR is particularly relevant in chronic inflammatory diseases such as asthma. Here weidentified that ORMDL3, the only genetic risk factor recently associated to asthma in a genome wide study,alters ER-mediated Ca21 homeostasis and facilitates the UPR. Heterologous expression of human ER-residenttransmembrane ORMDL3 protein increased resting cytosolic Ca21 levels and reduced ER-mediated Ca21 signal-ing, an effect reverted by co-expression with the sarco-endoplasmic reticulum Ca21 pump (SERCA). IncreasedORMDL3 expression also promoted stronger activation of UPR transducing molecules and target genes whilesiRNA-mediated knock-down of endogenous ORMDL3 potentiated ER Ca21 release and attenuated the UPR.In conclusion, our findings are consistent with a model in which ORMDL3 binds and inhibits SERCA resultingin a reduced ER Ca21 concentration and increased UPR. Thus, we provide a first insight into the molecular mech-anism explaining the association of ORMDL3 with proinflammatory diseases.

INTRODUCTION

The ER is essential for the generation of intracellular Ca2þ

signals, functioning as a regulated Ca2þ store (1,2). Thereare Ca2þ release channels (e.g. inositol trisphosphate receptorand ryanodine receptor) that control the exit of Ca2þ from theER into the cytoplasm and pumps [sarco-endoplasmic reticu-lum Ca2þ ATPase (SERCA)] that return Ca2þ to the ER. Inthis sense, the activity of SERCA determines the rate ofremoval of cytosolic Ca2þ, shaping the Ca2þ signal andhelping to maintain low cytosolic Ca2þ levels. SERCAactivity also influences Ca2þ-dependent cellular responses bydetermining the ER [Ca2þ] that is available for release inresponse to the next stimulus (2). Consistent with the keyrole of SERCA in Ca2þ homeostasis, several pathological con-ditions presenting ER Ca2þ dysregulation are associated withSERCA dysfunction (3), including asthma (4) and Alzheimer

disease (5,6), although in the latter case ER Ca2þ dysregula-tion has been also attributed to a Ca2þ leak via presenilin(7) or via the ryanodyne receptor (8).

The assembly and folding of numerous proteins also occursat the ER, a process that requires appropriate ER Ca2þ levels(9–11). Alterations of protein folding or Ca2þ levels withinthe ER result in the unfolded-protein response (UPR) (12–16). The UPR involves different signaling pathways thatsense conditions of ER stress and trigger the subsequent cellularhomeostatic response (17). Although the UPR has been charac-terized in different cell models, it is particularly relevant in cellsfrom the immune system where it is related to lymphocytedevelopment (18) as well as the generation and function ofantibody-secreting plasma cells (19). The UPR can alsotrigger the activation of NF-kB and JNK (20–22), key mol-ecules in the onset of inflammation. Together, the UPR hasbeen considered a condition closely related to the inflammatory

†The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

�To whom correspondence should be addressed at: Laboratory of Molecular Physiology and Channelopathies, Universitat Pompeu Fabra, Parc deRecerca Biomedica de Barcelona, Room 341, C/ Dr. Aiguader 88, Barcelona 08003, Spain. Tel: þ34 933160854; Fax: þ34 933160901; Email:[email protected]

# The Author 2009. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

Human Molecular Genetics, 2010, Vol. 19, No. 1 111–121doi:10.1093/hmg/ddp471Advance Access published on October 8, 2009

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response of specialized cells (17), either through the differen-tiation of inflammatory cells or the production of moleculesinvolved in the onset of inflammation (23).

Recently, the ORMDL3 gene, coding for the ER-residenttransmembrane protein ORMDL3 (24), has been associatedto asthma (25–29), a chronic inflammatory disease of theairways (30,31). ORMDL3 shows a wide distribution in differ-ent tissues, being particularly high its expression in cells par-ticipating in the inflammatory response (24,25). A common C/T polymorphism (rs7216389) controlling ORMDL3 expressionwas associated to asthma (25), but neither the function ofORMDL3 or the underlying mechanism for its associationwith asthma is known at present. Therefore, consideringthat: (i) there is a close relationship between UPR andinflammation; (ii) different transmembrane proteins of theER participate in the sensing and initiation of the UPR;(iii) perturbation of ER calcium stores induces the UPR and(iv) diminished SERCA expression contributes to airwayremodeling (4); we examined whether ORMDL3 may modu-late ER-mediated Ca2þ signals and/or participate in theUPR, thereby providing a plausible pathophysiological mech-anism explaining its association to asthma.

RESULTS AND DISCUSSION

Expression and topology of ORMDL3

Computer-based protein sequence analysis of the ER-residenthuman ORMDL3 protein originally predicted four membrane-spanning domains with different hydrophobicity scores (24).To obtain more detailed topological information, we used a flu-orescence protease protection assay (32). Three versions ofORMDL3 tagged with fluorescence proteins (FP) were gener-ated: at the N-terminus; C-terminus and amino acid 79 of theprotein (Fig. 1A). HEK293 cells were transfected with differentcombinations of ORMDL3, STIM1 (FP in its lumenalN-terminus) (33), DsRed-ER marker (FP in ER lumen) andTRPV4 (FP attached to the cytoplasmic C-terminus) (34)(Fig. 1A). Following permeabilization with 20 mM digitonin,images were obtained in the absence of trypsin (Fig. 1B). Additionof trypsin (Fig. 1C) resulted in rapid and complete loss of C- andN-tagged ORMDL3 signals as well as TRPV4 signal, indicatingthat ORMDL3 N- and C-terminus were facing the cytoplasm.In contrast, intrareticular fluorescence associated with STIM1and DsRed-ER did not change dramatically in the presence oftrypsin. Neither did the ORMDL3-generated fluorescence

Figure 1. Localization and topology of ORMDL3. (A) Schematic cartoon of YFP-tagged ORMDL3 constructs and cellular localization of different moleculeswith fluorescent protein (FP) tags. HEK293 cells co-expressing different combinations of ORMDL3-CFP or ORMDL3-YFP, ER targeting sequence of calreti-culin fused to Discosoma sp. red fluorescent protein, STIM1-YFP and TRPV4-CFP were treated with 20 mM digitonin. Images were taken before (B) and after(C) application of trypsin (2 mM). The FP color combination was selected to code cytosolic fluorescent markers in blue and intrareticular fluorescent markers inyellow for transmembrane proteins and red for globular proteins. (D) Schematic structure cartoon based on predictions obtained using TMHMM-2 (www.cbs.dtu.dk/services/TMHMM-2.0), PSIPRED V2.3 (bioinf.cs.ucl.ac.uk/psipred) and the topology experiments shown in a-c. Scale bars 10 mm.

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tagged at amino acid 79, suggesting that the central region ofORMDL3 faces the ER lumen. Based on these results and thefact that only two domains, of around 20 amino acids each (Sup-plementary Material, Fig. S1), show considerable hydrophobicityscores (.80% probability), we proposed a topology model forORMDL3 consisting of two transmembrane domains, with N-and C-terminus facing the cytoplasm and a large loop withinthe ER lumen (Fig. 1D).

Impact of ORMDL3 on cellular Ca21 homeostasis

The impact of heterologous expression of ORMDL3 onER-mediated intracellular Ca2þ signals was evaluated inHEK293 cells. Cytosolic Ca2þ signals (using fura-2 calcium sen-sitive dye) were recorded from GFP- and ORMDL3-transfectedcells in response to carbachol, a muscarinic agonist that triggersthe release of Ca2þ from inositol trisphosphate (IP3)-sensitive

Figure 2. Decreased ER Ca2þ signal in cells overexpressing ORMDL3. (A) Changes in cytosolic [Ca2þ]cyt (fura-2 ratio) evoked by 5 mM carbachol in Ca2þ-containing solutions. (B) ORMDL3 overexpression slows the clearance of cytosolic Ca2þ following carbachol stimulation in Ca2þ-free bath solutions. Tracesshow mean ratio normalized to peak Ca2þ signals in GFP and ORMDL3 transfected HEK293 cells (n . 50 for each condition). Time constants for Ca2þ clear-ance are 1.47 min and 3.7 min for GFP- and ORMDL3-transfected cells, respectively. (C and D), Changes in [Ca2þ]ER (mag fura-2 ratio) in GFP- (C) andORMDL3-transfected cells (D) exposed to 5 mM IP3. Second exposure to IP3 was followed by addition of 1 mM TG. (E) Mean values of basal [Ca2þ]ER

(mag fura-2 ratios). (F) Time course of Ca2þ reuptake into the ER in GFP- and ORMDL3-transfected HEK293 cells exposed to 5 mM IP3. Traces show normal-ized mean ratio Ca2þ signals in GFP and ORMDL3 transfected HEK293 cells. Time constants for Ca2þ reuptake are 15+2 min for GFP- (n ¼ 16) and 26+5 min (n ¼ 18) for ORMDL3-transfected cells. �P , 0.05 (versus GFP transfected cells), Student’s t-test.

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ER (Fig. 2A). The Ca2þ signal generated, which mainly reflectsthe amount of Ca2þ stored in the ER, was quantified by calculat-ing the mean area under the curve (inset). ORMDL3 overexpres-sion also slowed the decay rate of cytosolic Ca2þ clearancefollowing carbachol stimulation (Fig. 2B), indicative ofdecreased SERCA activity (5,35–37), as this Ca2þ pump is incharge of replenishing the stores with Ca2þ pumped from thecytosol into the ER (38). This observation was further confirmedby direct measurements of intra-ER Ca2þ levels using a low-affinity Ca2þ-sensitive dye (mag fura-2) trapped within the ERin cells permeabilized with digitonin (39). Higher resting magfura-2 ratios were observed in GFP- (Fig. 2C) than ORMDL3-transfected HEK293 cells (Fig. 2D). Mean basal ratios areshown in Fig. 2E. When permeabilized cells were stimulatedwith 5 mM IP3 the ratio decreased rapidly in both GFP- andORMDL3-transfected cells. Upon IP3 removal, ratio increased,indicating reuptake of Ca2þ into the IP3-sensitive store (Fig. 2Cand D), but with a slower kinetics in ORMDL3-cells. Exponentialfitting of Ca2þ reuptake gave mean time constants of 15+2 min(n ¼ 16) and 26+5 min (n ¼ 18) for GFP- and ORMDL3-expressing cells (P , 0.05), respectively (Fig. 2F). Addition ofthe SERCA inhibitor thapsigargin (TG) (3,38) prevented Ca2þ

reuptake into the intracellular organelle following a second IP3

challenge (Fig. 2C and D). This approach using mag fura-2 thatonly reports on Ca2þ signal within the ER, minimized the effectof Ca2þ extrusion through the plasma membrane on calciumclearance that may be present in the experiment shown inFig. 2B. In other words, while in Figure 2B, time constant ofCa2þ clearance from the cytosol may be influenced by bothCa2þ reuptake into the ER and Ca2þ extrusion through theplasma membrane, data shown in Figure 2F only refers to thetime constants of Ca2þ reuptake into the ER.

The effect of other stimuli and knockdown of endogenousORMDL3 with siRNA was also tested. ORMDL3 overexpres-sion decreased Ca2þ response to the SERCA inhibitor cyclo-piazonic acid (CPA, 10 mM), which promotes passive releaseof Ca2þ from ER (3,38), whereas ORMDL3 siRNA poten-tiated the response to CPA (Fig. 3B), recorded in theabsence of extracellular Ca2þ to avoid possible contributionof Ca2þ influx across the plasma membrane. Mean curveareas obtained following CPA treatment in cells transfectedwith GFP, control siRNA, ORMDL3 and ORMDL3 siRNAare shown in Figure 3C. The fact that similar effectwas observed in ORMDL3-transfected cells exposed to anIP3-generating stimulus and the SERCA inhibitor CPA,suggests that the effect shown in Figure 2A is mainly producedby inhibition of Ca2þ reuptake into the ER, rather thaninhibition of the IP3 receptor. Although, at present, wecannot unequivocally discard the latter possibility.

Note the inverse correlation between ORMDL3 levels andCPA-induced, ER-mediated Ca2þ response: the highest Ca2þ

signal corresponded with lowest ORMDL3 levels. On theother hand, a positive correlation was detected betweenbasal cytosolic Ca2þ concentration and ORMDL3 levels(Fig. 3D). Similar results were obtained with ionomycin, anionophore that releases Ca2þ from most intracellular storesin the absence of external Ca2þ (Fig. 3E). Overexpression ofORMDL3 was always associated with a significant reductionin the ER Ca2þ response while knocking down ORMDL3increased such response.

ORMDL3 interacts with and modulates SERCA activity

ORMDL3-GFP co-localized with endogenous SERCA2b inHEK293 cells (Fig. 4A) and SERCA2b (either native or overex-pressed) co-immunoprecipitated ORMDL3-GFP in HEK293cells (Fig. 4B). Expression of the proteins of interest wasprobed by western blotting in the same cell lysates (input) usedfor immunoprecipitation (Supplementary Material, Fig. S2).These results suggested that the modulatory effect ofORMDL3 upon SERCA may involve a direct association. Aswith other SERCA-interacting proteins that regulate pumpactivity (40–42), co-immunoprecipitation of ORMDL3 andSERCA2b was stronger at 5 mM Ca2þ.

Next, we attempted reverting ORMDL3-induced phenotypeby co-expressing SERCA and ORMDL3. Cells overexpressingORMDL3 and stimulated with 1 mM TG—that also promotespassive release of Ca2þ from ER—showed reduced Ca2þ

release from the ER, a phenotype that was reverted byco-expressing SERCA2b (Fig. 5A and B). Basal cytosolic[Ca2þ] was also returned to control conditions in cellsco-expressing SERCA2b and ORMDL3 (61+1 nM, n ¼ 80;P ¼ 0.1 versus GFP transfected cells).

Our results showed: lower ER Ca2þ levels and release fromthe ER; slower Ca2þ reuptake into the ER and cytosolic Ca2þ

clearance; and higher basal cytosolic Ca2þ concentrations.Besides, co-localization and co-immunoprecipitation of bothORMDL3 and SERCA suggest a physical interactionbetween both proteins. Together, these results obtained fromORMDL3 overexpressing cells are consistent with the inhi-bition of the SERCA pump that contributes to maintain lowcytosolic Ca2þ concentrations by pumping Ca2þ from thecytosol into the ER (3,38). Although at present we cannotfully discard that ORMDL3 may also present certain channelactivity, as reported for other modulators of SERCA (7).

In our attempt to identify ORMDL3 functional motifs, wedeleted the last nine amino acids of ORMDL3 that contain aputative ER retention sequence (Fig. 5C). ORMDL3-D145–153 showed cellular distribution similar to ORMDL3-WT(Supplementary Material, Fig. S3) but lost the inhibitoryeffect on ER-mediated Ca2þ signaling, evaluated byexposing cells to 5 mM carbachol (Fig. 5C and D). Interest-ingly, ORMDL3-D145–153 did not lose the ability toimmunoprecipitate with SERCA (results not shown),suggesting a separation between the ORMDL3 domainsinvolved in functional and physical interaction with SERCA.

ORMDL3 facilitates UPR

A decrease in ER Ca2þ (using SERCA inhibitors) triggers theUPR, characterized by activation of signaling molecules and, ulti-mately, increased transcriptional activation of immediate-earlygenes and others directly related with the onset of inflammation(17). UPR signaling pathways at the ER usually follow theactivation of one or more of the known protein sensors: pancreaticendoplasmic reticulum kinase (PERK), inositol-requiring 1 a(IRE1a) or activating transcription factor 6 (ATF6). The levelsof ER stress were evaluated by analyzing the phosphorylationof eukaryotic initiation factor 2a (eIF2a), an early markerof UPR downstream of PERK activation (15,17). ORMDL3overexpression induced significantly higher basal eIF2a


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phosphorylation, without changing total eIF2a, whereasORMDL3 knock-down with siRNA significantly reduced thephosphorylated eIF2a levels (Fig. 5E and F). Overexpression ofORMDL3-D145–153 did not significantly affect eIF2a phos-phorylation (Fig. 4E and F). Therefore, expression of functionalORMDL3 correlated with the level of phosphorylated eIF2a.We also evaluated the influence of ORMDL3 on two UPRtarget genes downstream of eIF2a, BIP and EGR-1 (15,43).

Similar to what we observed with eIF2a, overexpression ofORMDL3 augmented BIP while overexpression ofORMDL3-D 145–153 did not significantly affect BIP expression(Fig. 5G). Loss of ORMDL3—with siRNA—also attenuated BIPexpression (Supplementary Material, Fig. S4A). Similar resultswere obtained when evaluated EGR-1 transcription (Supplemen-tary Material, Fig. S4B). Typically, ER stress activates differentUPR signaling pathways (22), although preferences may exist

Figure 3. Silencing ORMDL3 in HEK293 cells increases ER Ca2þ signals. (A) Conventional (top) and quantitative RT–PCR (bottom) demonstrating down-regulation of ORMDL3 in HEK293 cells transfected with ORMDL3 siRNA, relative to control siRNA transfected cells (H2O: no template control).(B) Genetic down-regulation of ORMDL3 expression with ORMDL3 siRNA (filled triangle) increases ER Ca2þ response to cyclopiazonic acid (CPA,10 mM) while ORMDL3 overexpression (open circle) lowers ER Ca2þ response compared with GFP transfected cells (filled circle). (C) Average of areasunder the curves derived from the experiment in (B). Mean response to control siRNA (not plotted in B, for the sake of clarity) is also included. (D) Meanvalues of basal cytosolic [Ca2þ]. (E) ORMDL3 siRNA (filled circle) increases ER Ca2þ response to ionomycin (1 mM) while ORMDL3 overexpression(open circle) lowers ER Ca2þ response compared to GFP transfected cells (filled circle). Inset, mean average of areas under the curves. Data are expressedas the mean+SEM (N values shown for each bar). �P , 0.05 (versus GFP transfected cells), one way ANOVA and Tukey post hoc for comparison of multipleconditions or Student’s t-test for comparison of two conditions.


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depending on the UPR originating condition. To test whetherORMDL3 influences other PERK- and eIF2a-independent UPRsignaling pathways, we evaluated Xbp-1 mRNA splicing byreverse transcription polymerase chain reaction in HEK-293cells, a process that occurs following IRE1a activation. Overex-pression of ORMDL3 did not affect Xbp-1 mRNA splicingeither under basal conditions or following TG treatment (Sup-plementary Material, Fig. S4C), suggesting that the main impactof ORMDL3 is on PERK/eIF2a pathway.

Together, these results are consistent with the participationof ORMDL3 in the development of the UPR, a process thatcan initiate inflammation and may explain the reported associ-ation of ORMDL3 with asthma. In the chronic inflammatoryfeature of asthma, T cells are pivotal initiators or mediatorsof this response (31). Consequently, we assessed the impactof ORMDL3 on calcium homeostasis and UPR in the humanT-cell line Jurkat, a model widely used in the study ofT-cell signaling. Similar to the reported effect on HEK293

cells, ORMDL3 overexpression in Jurkat cells decreased theCa2þ response to the SERCA inhibitor CPA (30 mM)(Fig. 6A). Phosphorylation of eIF2a was also evaluated inJurkat cells transfected with CFP or ORMDL3-CFP. Due tothe low transfection rate of Jurkat cells, eIF2a-P was analyzedon individual cells by immunofluorescence confocalmicroscopy (Fig. 6B). Mean normalized levels of eIF2a-Pand percentage of responding Jurkat cells are clearly increasedin ORMDL3-transfected cells, compared with CFP transfectedJurkat cells (Fig. 6C and D).

Conclusions

Our data reports on the involvement of ORMDL3 onER-mediated Ca2þ signaling and facilitation of ER-mediatedinflammatory responses. Besides, our study increases theunderstanding of the cellular and molecular mechanismsunderlying the reported association of ORMDL3 with inflam-matory diseases such as asthma (25) and Crohn’s disease (44).Interestingly, the increased risk of asthma conferred byORMDL3 variants has been recently associated to tobaccosmoke (29), being this environmental disease modifier aninducer of UPR (45). In conclusion, our observation offers anovel target for the study of ER Ca2þ signaling and itsimpact on disease pathophysiology.

MATERIALS AND METHODS

Plasmids and cell transfection

Human ORMDL3 and pig SERCA2b expression vectors werea kind gift from Drs R. Gonzalez-Duarte (University of Barce-lona) and M. Brini (University of Padova), respectively;pDsRed-ER was obtained from CLONTECH. ORMDL3tagged with CFP at the N- and C-terminus was generated bysubcloning ORMDL3 cDNA into pCDNA3-CFP andpECFP-C1 vectors, respectively. ORMDL3 tagged with YFPat amino acid position 79 and ORMDL3-D1452153 (deletionfrom amino acid 145 to the C-terminus) were generated byPCR. All constructs were verified by sequencing (Big Dye3.1, AbiPrism, Applied Biosystems).

HEK293 cells were transiently transfected with ExGen500(Fermentas MBI,) following manufacturer instructions.Jurkat cells were transfected in 24-well plates using 1 mgplasmid DNA and 2 ml TransIT-Jurkat Transfection Reagent(Mirus Bio Corporation) per well.

Figure 4. ORMDL3 interacts with SERCA. (A) Colocalization of ORMDL3and SERCA2b in HEK293 cells transfected with ORMDL3-GFP.ORMDL3-GFP signal (green) and SERCA2b (1:250) immunostaining in per-meabilized cells (red). Merge images appear yellow in colocalization areas.Scale bar 20 mm. (B) Co-immunoprecipitaion of SERCA2b and ORMDL3in HEK293 cells transfected with ORMDL3-GFP (lane 2) or ORMDL3-GFPþSERCA2b (lanes 1 and 3–5) was analyzed under different conditions: controlnominal free calcium (lane 3), 5 mM Ca2þ (lane 4) or 5 mM EGTA (lane 5).Immunoprecipitation lanes in the absence (lane 1) or presence ofa-SERCA2b polyclonal antibody (lanes 2–5). Immunocomplexes were ana-lyzed by western blots with anti-GFP (bottom) to detect ORMDL3. Meanimmunoprecipitated signals are shown for each lane (n ¼ 3).

Figure 5. ORMDL3 modulates SERCA activity and UPR. (A) ER Ca2þ release evoked by 1 mM TG in Ca2þ-free solutions in HEK293 cells transfected with GFP(filled circle), ORMDL3 (open circle) or ORMDL3þSERCA (filled square). (B) Average ER Ca2þ release (area under the curve) obtained by integrating Ca2þ

signals of individual cells. (C) Schematic structure of ORMDL3-D145–153 construct and ER Ca2þ release evoked by 5 mM carbachol in Ca2þ-free solutions inHEK293 cells transfected with GFP (filled circle) or ORMDL3 (open circle) or ORMDL3-D145–153 (filled circle). (D) Average ER Ca2þ release (area under thecurve) obtained by integrating Ca2þ signals of individual cells. Data are expressed as the mean+SEM (N values shown for each bar). (E) Total and phosphory-lated eIF2a were detected by western blot analysis of whole extracts from EGFP, ORMDL3, ORMDL3 siRNA and ORMDL3-D145–153 transfected cells. (F)Mean relative levels of phosphorylated eIF2a/total eIF2a obtained from four independent experiments. (G) Messenger RNA levels of BIP (relative to Rlp19)were analysed by quantitative RT–PCR in cells transfected with GFP and ORMDL3 and ORMDL3-D145–153. Data are expressed as the mean+SEM(n values shown for each bar). �P , 0.05 (versus GFP transfected cells), one way ANOVA and Tukey post hoc for comparison of multiple conditions.


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Measurement of intracellular [Ca21]

Cytosolic Ca2þ signal was determined at RT in cells loadedwith 4,5 mM fura-2.AM (20 min) as previously described

(46). Cytosolic [Ca2þ] increases are presented as the ratio ofemitted fluorescence (510 nm) after excitation at 340 and380 nm, relative to the ratio measured prior to cell stimulation

Figure 6. Effect of ORMDL3 on T cell ER Ca2þ signaling and stress. (A) Changes in cytosolic Ca2þ evoked by 30 mM CPA in Jurkat cells overexpressing CFP(filled circle) or human ORMDL3-CFP (open circle). Insets show average [Ca2þ] increases (Area) obtained by integrating the Ca2þ signals of individual cells.(B) Confocal CFP (left) and phosphorylated eIF2a (right) immunofluorescence images of Jurkat cells transfected with CFP or human ORMDL3-CFP. Arrowsmark CFP and eIF2a-P signals in the same cell. (C) eIF2a-P fluorescence signal normalized by area in non-transfected cells (NT), CFP and ORMDL3-CFPtransfected cells. Data are expressed as the mean+SEM (N values shown for each bar). �P , 0.05 (versus non-transfected cells), one way ANOVA andTukey post hoc. CFP versus ORMDL3 P , 0.05. (D) Percentage of Jurkat cells showing increased eIF2a-P signal (.30% of the signal detected in NTcells). Number of positive cells over total shown for CFP and ORMDL3-CFP transfected cells. Scale bars 20 mm.


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(fura-2 ratio 340/380). Absolute basal Ca2þ concentration wasobtained from the fluorescence ratios using an on-cell cali-bration protocol (47). All experiments were carried out atroom temperature and cells were bathed in an isotonic solutioncontaining (in mM): 140 NaCl, 5 KCl, 1.2 CaCl2, 0.5 MgCl2, 5glucose, 10 HEPES (300 mosmol/l, pH 7.4 with Tris).Ca2þ-free solutions were obtained by replacing CaCl2 withequal amount of MgCl2 plus 0.5 mM EGTA.

To measure free [Ca2þ] inside the ER cells were incubated45 min at 378C with the low-affinity Ca2þ dye mag fura-2-AM(5 mM) in isotonic medium containing 0.02% pluronic F-127(39). To wash out the cytosolic dye, plasma membrane waspermeabilized in ATP-free intracellular-like media (in mM:120 KCl, 25 NaCl, 0.1 MgCl2, 0.75 CaCl2, 0.5 EGTA(�100 nM [Ca2þ]) and 10 HEPES; pH 7.2 with KOH,300 mOsm) containing 8 mM digitonin. Following permeabili-zation, digitonin was removed and 1 mM ATP added to theintracellular solution for at least 15 min, until stable magfura-2 340/380 ratio levels were obtained, which are pro-portional to [Ca2þ]ER.

Fluorescence protease protection assay

Twenty-four hours after transfection, live cells were permeabi-lized with 20 mM digitonin at RT for 3 min. After permeabili-zation, we incubated the cells with 2 mM trypsin for 2 min.The subcellular localization of tagged proteins was analyzedbefore and after trypsin treatment under a 40 � 1.32 Oil Ph3CS objective, LCS Leica Confocal software and Argon(488 nm, JDS Uniphase Corporation) and HeNe (555 and633 nm, JDS Uniphase Corporation and LASOS LasertechnikGmbH, respectively) lasers using an inverted Leica SP2 Con-focal Microscope, as previously described (32). Images weretaken at room temperature and were not further processedexcept to adjust brightness, contrast and color balance.

Expression knock-down and quantitativeRT–PCR analysis

Cells were seeded in 6-well plates at 90% confluency andexposed to 100 pmoles of ORMDL3 siRNA (50-TAAGTACGACCAGATCCATTT-30) (Qiagen) or control siRNA(50-AATTCTCCGAACGTGTCACGT-30) (Qiagen) dilutedinto 100 ml serum-free medium. Cells were transfected by aLipofectamine 2000 (Invitrogen) procedure following themanufacturer’s instructions, as described previously (48).RNA extraction (Nucleospin RNA II kit, Macherey-Nagel)was carried out 48 h after transfection and RT–PCR was per-formed as described previously (48) using SuperScrip-RTsystem (Invitrogen) and aliquots of 1 mg cDNA were usedas template for quantitative PCR. Quantitative RT–PCR wasperformed on an ABI Prism 7900HT (Applied Biosystems)with SYBR-Green (SYBR-Green Power PCR Master Mix,Applied Biosystems) and ORMDL3 QuantiTect PrimerAssay (Qiagen). Other primers used included: BiP 50-CGGGCAAAGATGTCAGGAAAG-30 and 50-TTCTGGAACGGGCTTCATAGTAGAC-30; EGR-1 50CAGCACCTTCAACCCTCAG-30 and 50-AGCGGCCAGTATAGGTGATG-30. Beta-actin or ribosomal protein Rpl19 served as an internal normaliza-tion standard (20). PCR conditions for ORMDL3 and EGR-1

were: 958C for 5 min; 958C for 30 s; 608 for 30 s, 728C for 30 s;728C for 5 min; with 40 cycles of amplification. PCR conditionsfor BIP were: 958C for 5 min; 958C for 30 s; 558 for 30 s, 728Cfor 1 min; 788C for 10 s; 728C for 5 min, with 40 cycles ofamplification.

Immunoprecipitation assay and immunodetection

Co-immunoprecipitation experiments were run as previouslydescribed (46). HEK293 cells were transiently transfectedwith human ORMDL3-GFP (or ORMDL3:ORMDL3-GFPratio 1:1) and pig SERCA2b plasmids were lysated withimmunoprecipitation buffer (0.5% Triton plus protease inhibi-tor cocktail in nominal free Ca2þ HBS or HBS containing1 mM TG, 5 mM Ca2þ or 5 mM EGTA) and centrifuged at100 000g to collect total protein in supernatant. Then1000 mg total protein were incubated at 48C overnight withanti-SERCA2b antibody (Abcam). After 2 h incubation with30 ml of G protein (Amersham) at RT, immunocomplexeswere washed with immunoprecipitation buffer five times andboiled for 5 min with loading buffer. Co-immunoprecipitationof ORMDL3 was detected with anti-GFP antibody (1:1000,mouse monoclonal antibody, Clontech Laboratories, Inc.).For western blotting of eIF2a, 50 mg of total protein obtainedfrom HEK293 cells 48 h after transfection with GFP,ORMDL3, ORMDL3-D145–153 or ORMDL3 siRNA wereseparated on 4–10% gradient polyacrylamide gel and trans-ferred to PVDF membranes. Primary antibodies used weremouse anti-eIF2a (1:500) and rabbit anti-phosphoS51-eIF2a(1:500) from Abcam. Secondary antibodies used were horse-radish peroxidase-conjugated anti-mouse and anti-rabbit IgG(1:3000, GE Healthcare). Immunoreactive signal was detectedby SuperSignal West Pico Chemiluminiscent substrate(Pierce) and visualized by Molecular Imager Chemidoc XRSsystem (Biorad).

For immunodetection of phoshorylated eIF2a, 24 h aftertransfection of Jurkat cells with ORMDL3-CFP or pCFPvectors, cells were attached for 30 min to poly-L-Lysinecoated coverslips, fixed with 4% PFA, permeabilized with0.1% Trinton x-100 in PBS and blocked with 1% BSA, 2%FBS, 0.05% Triton in PBS. Samples were stained forphospho-eIF2a using anti-phosphoS51-eIF2a antibody (1:50in blocking solution). Secondary antibody was a goat anti-rabbit Alexa Fluor555 (Molecular Probes). Images wereacquired using an inverted Leica SP2 Confocal Microscopewith a 40 � 1.32 Oil Ph3 CS objective and analyzed usingImageJ software. Intensity/area ratio of every transfected cellwas normalized to the intensty/area mean of the surroundingnon transfected cells in the same image. EndogenousSERCA immunodetection was carried out following thesame procedure using SERCA 2b antibody (1:250, Genetex).

Statistics

All data were expressed as means+SEM of N (numberof cells analyzed) or n (number of experiments carried out).Statistical analysis was performed with Student’s unpairedtests, or one-way analysis of variance (ANOVA) using Sigma-Plot or OriginPro software. Bonferroni’s or Tukey’s tests were


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used for post hoc comparison of means. The criterion for asignificant difference was a final value of P , 0.05.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

ACKNOWLEDGEMENTS

We thank C. Plata for technical support, R. Gonzalez-Duarte(University of Barcelona) for ORMDL3 plasmid, M. Brini(University of Padova) for SERCA2b plasmid andS. Muallen (University of Texas) for STIM1 plasmid.

Conflict of Interest statement. None declared.

FUNDING

This work was supported by Spanish Ministry of Science andInnovation (SAF2006-04973, SAF2009-09848); Fondo deInvestigacion Sanitaria (Red HERACLES RD06/0009); Gen-eralitat de Catalunya (SGR05-266); and Fundacio la Maratode TV3 (061331 and 080430). M.A.V. is the recipient of anICREA Academia Award.

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ORMDL3 modulates store-operated calcium entry and lymphocyte activation

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