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Cell Calcium 42 (2007) 83–90

Calumin, a novel Ca2+-binding transmembrane proteinon the endoplasmic reticulum

Miao Zhang a,b,1, Tetsuo Yamazaki c,1, Masayuki Yazawa a,b, Susan Treves d, Miyuki Nishi a,b,Machiko Murai b, Eisuke Shibata b, Francesco Zorzato d, Hiroshi Takeshima a,b,∗

a Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japanb Department of Medical Chemistry, Graduate School of Medicine, Tohoku University, Miyagi 980-8575, Japan

c The 21st Century Center of Excellence Program, Graduate School of Medicine, Tohoku University, Miyagi 980-8575, Japand Department of Anesthesiology and Research, University of Basel Kantosspital, Basel, Switzerland

Received 21 June 2006; received in revised form 10 October 2006; accepted 22 November 2006Available online 3 January 2007

bstract

We have identified a novel endoplasmic reticulum (ER)-resident protein, named “calumin”, which is expressed in various tissues. Thisrotein has a molecular mass of ∼60 kDa and is composed of an ER-luminal domain rich in acidic residues, a single transmembrane segment,nd a large cytoplasmic domain. Biochemical experiments demonstrated that the amino-terminal luminal domain is capable of bindinga2+ with a high capacity and moderate affinity. In embryonic fibroblasts derived from calumin-knockout mice exhibiting embryonic and

2+ 2+ 2+

eonatal lethality, fluorometric Ca imaging detected insufficient Ca contents in intracellular stores and attenuated store-operated Cantry. Moreover, the mutant fibroblasts were highly sensitive to cell death induced by ER stress. These observations suggest that calumin playsn essential role in ER Ca2+ handling and is also implicated in signaling from the ER, which is closely associated with cell-fate decision.

2006 Elsevier Ltd. All rights reserved.

out mo

eywords: Ca2+-binding protein; ER stress; Intracellular Ca2+ store; Knock

Abbreviations: ATF, activation of transcription factor; [Ca2+]i, intra-ellular [Ca2+]; CNX, calnexin; CBB, Coomassie brilliant blue; CPA,yclopiazonic acid; CRT, calreticulin; eIF, eukaryotic translation initiationactor; ER, endoplasmic reticulum; GST, glutathione S-transferase; IRE,nositol-requiring transmembrane kinase; JNK, c-Jun N-terminal kinase;

Ab, monoclonal antibody; MEF, mouse embryonic fibroblast; NFAT,uclear factor of activated T cells; PERK, protein kinase like ER kinase;CR, polymerase chain reaction; PDI, protein disulfide isomerase; SERCA,arcoplasmic/endoplasmic Ca2+-Mg2+ ATPase; STS, staurosporine; SR, sar-oplasmic reticulum; TRAF, tumor necrosis factor-associated factor; TG,hapsigargin; TNF, tumor necrosis factor; TM, tunicamycin; UPR, unfoldrotein response; xbp, X-box-binding protein∗ Corresponding author at: Department of Biological Chemistry, Gradu-te School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501,apan. Tel.: +81 75 753 4572; fax: +81 75 753 4605.

E-mail address: takeshim@pharm.kyoto-u.ac.jp (H. Takeshima).1 Contributed equally to this work.

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use; Store-operated Ca2+ entry

. Introduction

The endoplasmic/sarcoplasmic reticulum (ER/SR) is aultifunctional organelle and governs a wide variety of cellu-

ar processes through its Ca2+ storage capability [1–3]. TheR plays a major role in protein synthesis and a constanta2+ level in the lumen is essential for protein folding androcessing. Ca2+ handling by the ER/SR is constituted ofhree major aspects: Ca2+ uptake mediated by Ca2+-Mg2+

TPase (SERCA), Ca2+ storage utilizing various luminala2+-binding proteins, and Ca2+ release through inositol,4,5-trisphosphate or ryanodine receptors. Recently studiesave identified two major families of ER-resident Ca2+-inding proteins with putative roles for fundamental cellular

vents including protein folding and intracellular Ca2+ signal-ng. The calreticulin (CRT)/calnexin (CNX) family proteinsind Ca2+ via acidic amino acid clusters [4], while the CRECCab45/reticulocalbin/ERC45/calumenin) family members

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hare multiple EF-hand motifs [5]. Although CRT/CNX fam-ly members seem to function as molecular chaperones, thehysiological roles of the CREC family are largely unknown.

number of Ca2+-binding proteins localized in the ERemain uncharacterized as several new members have beeneported in recent years [6–8].

In addition to housekeeping functions, the ER also plays aentral role in cellular signaling pathways inducing store-perated Ca2+ entry (SOCE) [9] and ER-stress responses10]. Ca2+ release and the resulting Ca2+ depletion in intra-ellular stores triggers cell-surface Ca2+ influx, and resultingOCE activates a series of cellular processes such as geneegulation, cell division and apoptosis. Meanwhile, Ca2+

tore depletion induces ER-stress responses because cor-ect protein folding and processing require Ca2+-dependenteactions [11] and structurally defective proteins extensivelyccumulate under store-depleted conditions. The cellularesponses, collectively referred to as the “unfolded proteinesponse (UPR)”, operate to augment the capacity of theR to refold and degrade aberrant proteins. These protec-

ive mechanisms in the ER seem to ensure multifaceted Ca2+

unctions. Towards the understanding of such physiologicalignaling, it is important to identify and characterize signalingroteins transmitting ER information to the cytoplasm.

In this work, we report a novel ER transmembrane pro-ein named calumin. On the basis of the results obtained, weropose that calumin contributes to Ca2+ storage functions,OCE, and ER stress responses in a wide variety of cell types.

. Materials and methods

.1. Reagents

Thapsigargin (TG), tunicamycin (TM), and cyclopiazoniccid (CPA) were from Sigma (St. Louis, MO). StaurosporineSTS) was from Wako (Osaka, Japan). Tumor necrosisactor-� (TNF-�) was from Peprotech (Calabash, NC). Thentibodies used for immunoblotting were as follows: antibod-es against CNX, G�q/11, CRT, SERCA type-2, CHOP, andaspase-3 (Santa Cruz Biotech. Santa Cruz, CA), antibod-es against phospho-eukaryotic initiation factor 2� (eIF2�),-Jun N-terminal kinase (JNK), phospho-JNK, caspase-9,aspase-12, and cleaved caspase-3 (Cell Signaling Tech-ology, Danvers, MA), antibodies against GM130 and BiPBD Biosciences, Franklin Lakes, NJ), anti-cytochrome oxi-ase subunit IV (COX4) antibody (Clontech, Mountainiew, CA), anti-eIF2� antibody (Abcam, Cambridge, UK),nti-FLAG M2 antibody (Sigma), and anti-protein disulfidesomerase (PDI) antibody (Stressgen, Victoria, BC, Canada).

.2. cDNA cloning and recombinant protein expression

In a survey of novel proteins from the mouse brainsing the signal sequence trap method [12,13], we isolatedpartial calumin cDNA fragment containing the 5′-flanking

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m 42 (2007) 83–90

equence and protein-coding sequence for an amino-terminalegion. Database searches using the cDNA sequence thusetermined found the mouse full-length EST clones encod-ng calumin (GenBank accession numbers BY711094 andB040489) and the reported sequences were confirmed inur cDNA cloning. Total RNA samples were prepared fromdult C57BL/6J mouse tissues and Northern blot analysis wasarried out as described previously [14].

Calumin cDNA fragments for the luminal domain (aminocid residues 19–135) and cytoplasmic domain (residues56–483) were amplified using polymerase chain reactionPCR) and cloned into the pGEX-6P-1 vector (GE Health-are, Piscataway, NJ). Glutathione S-transferase (GST)usion proteins were purified from bacterial cultures usinglutathione-Sepharose 4B (GE Healthcare) according to theanufacturer’s instruction and subjected to Ca2+-binding

nalysis. 45Ca2+ overlays on blotting sheets were carried outs described previously [15]. 45Ca2+ equilibrium binding waserformed using a continuous flow microdialysis chamber asescribed previously [16].

The FLAG-tagged calumin cDNAs were PCR-generatednd cloned into the pcDNA3.1+ vector (Invitrogen).EK293 cells cultured on collagen-coated glass slidesere transfected with the expression plasmids together withDsRed2-ER or pDsRed2-Mito (Clontech). At 36 h post-ransfection, the cells were fixed with 4% paraformaldehyde,ermeabilized using 0.1% Triton X-100 in PBS, preblockedith 5% BSA in PBS, and reacted with the anti-FLAG2 antibody (Sigma). Immunoreactivity was visualized by

ITC-conjugated rabbit anti-mouse IgG (Dako, Glostrup,enmark), and observed using a confocal microscope (modelX51TRF, Olympus, Tokyo, Japan).

The membrane topology of calumin was examined asssentially described previously [17]. Briefly, HEK293 cellsere transfected with the FLAG-tagged calumin cDNAs,arvested at 36 h post-transfection, resuspended in 1.6 mlf buffer A (10 mM Hepes-KOH (pH 7.4), 10 mM KCl,.5 mM MgCl2, 5 mM sodium EDTA, 5 mM sodium EGTAnd 250 mM sucrose), and homogenized with a dounce tis-ue grinder. After the removal of cell debris by low-speedentrifugation (1000 × g for 5 min), the homogenate wasentrifuged at 20,000 × g for 15 min to recover membraneesicles. The resulting membrane pellets were resuspendedn buffer A containing 100 mM NaCl, and aliquots of thereparations (30 �g) were treated with varying concentra-ions of trypsin for 30 min at 30 ◦C in the absence or presencef 1% Triton X-100. The reactions were stopped by addingoybean trypsin inhibitor (Sigma) at a final concentration of0 units/�l and the degradability of recombinant proteins wasnalyzed by immunoblotting.

.3. Immunoblot analysis

Wistar rats were immunized with GST recombinant pro-ein carrying the calumin cytoplasmic domain (residues56–483) and hybridoma cells were produced by fusion of the

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ymphocytes with NS-1 cells [14]. Of the monoclonal anti-odies (mAbs) derived from several positive clones, mAb29eacted specifically with mouse calumin. Biochemical sub-ellular fractionation of mouse cerebellum was performeds reported previously [18] and the resulting fractions werenalyzed by immunoblotting. Mouse embryos (embryonicay 14.5) were dissociated, minced and trypsinized to yieldultured mouse embryonic fibroblasts (MEFs). MEFs werearvested and lysed in a buffer (1% NP-40, 50 mM Tris-HClpH 7.5), 150 mM NaCl, and 4 mM EDTA) supplementedith protease inhibitors. Protein contents were determinedy the BCA Protein Assay Kit (Pierce, Rockford, IL). Formmunoblotting, equal amounts of proteins were separatedn SDS-polyacrylamide gels and transferred to polyvinyli-ene difluoride membranes (Millipore, Bedford, MA). Afterlocking, filters were probed with specific antibodies and theneacted with horseradish peroxidase-conjugated secondaryntibody. Immunoreactivity was visualized by an enhancedhemiluminescence detection system (GE Healthcare).

.4. Generation of knockout mice

A mouse genomic DNA library (Stratagene, La Jolla, CA)as screened with a calumin cDNA fragment as a hybridiza-

ion probe to yield phage clones carrying the 5′-terminalegion of the calumin gene. The generation of calumin-nockout mice was carried out as described previously [19].he targeting vector was constructed with the genomic DNA

ragments, the neomycin-resistance gene from pMC1 NeoStratagene), the diphtheria toxin gene from pMC1-DT-A,nd pBluescript SK (-) (Stratagene). The linearized vectoras transfected into embryonic stem cells and several clones

rom the resulting G418-resistant cells were shown to carryhe expected homologous mutation by Southern blot analy-is. Chimeric mice generated with the positive clone wererossed with C57BL/6J mice and transmitted the mutantene to their pups. The calumin-knockout and wild-typembryos obtained by crossing the heterozygous mutants weresed for the analysis in this study. To determine the mouseenotypes, PCR analysis was conducted using the followingrimers: CSN2 (5′-CACTCGCTGAGGCGACTCCGTAAC-′) and CSN3 (5′-CACGCATGTGCAAGGGTCGTTCAC-′). Total microsomal preparations from the neonatal heartsere analyzed with mAb29.

.5. Fluorometric Ca2+ imaging

MEFs were generally maintained in DMEM supple-ented with 10% fetal bovine serum. For Ca2+ imaging,EFs were plated onto glass-bottomed dishes (MatTek, Ash-

and, MA), grown for 24 h, and loaded for 60 min with�M Fura 2-AM (Dojindo, Kumamoto, Japan). MEFs were

ashed three times with a physiological salt solution (PSS:50 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2,.6 mM glucose, and 5 mM Hepes, pH 7.4) and incubatedor 20 min at room temperature before imaging. A cooled

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m 42 (2007) 83–90 85

CD camera mounted on a microscope equipped with a poly-hrometer (Meta Fluor Imaging System, Universal Imaging,awthorn, NY) was used to capture fluorescence imagesith alternating excitation at 340 and 380 nm and emission

t >510 nm.

.6. ER stress-related assays

For the annexin V-binding assay, MEFs were plated on2-well plates (7.5 × 104 cells per well) for 12 h and thenncubated for 24 h in the presence of TG, TM, STS orNF-� plus cycloheximide (10 �g/ml). MEFs were washedith phosphate-buffered saline, harvested, exposed to FITC-

onjugated annexin V and 7-amino-actinomycin D (BDiosciences), and analyzed by a FACSCalibur flow cytome-

er (BD Biosciences) as described in the instruction manual.or analyzing X-box-binding protein 1 (xbp1) RNA splic-

ng, first-strand cDNA was synthesized with Superscript IIeverse transcriptase (Invitrogen, Carlsbad, CA) using totalNA from MEFs as a template. Partial xbp1 cDNAs weremplified by PCR using the following primers: mXBP1-(5′-ACACGCTTGGGAATGGACAC-3′) and mXBP1-AS

5′-CCATGGGAAGATGTTCTGGG-3′), and analyzed ongarose gels.

.7. Retrovirus infection

The DNA fragments encoding C-terminally tagged wild-ype or truncated calumin were PCR-generated and clonednto the pQCXIP retroviral vector (Clontech). Recombinantirus was generated according to the manufacturer’s instruc-ion. MEFs (∼3 × 105 cells) were trypsinized, resuspendedith 2 ml of virus-containing precleared supernatant fromcoPack2-293 packaging cells (Clontech) and incubated onetroNectin dishes (Takara, Shiga, Japan) for 6 h. After aedium change to remove the virus-containing supernatant,EFs were cultured for ∼24 h and reseeded onto glass-

ottomed dishes. After additional 24 h culture, the cells wereubjected to fluorometric Ca2+ imaging and immunoblotting.

. Results

.1. Cloning of calumin

In our attempt to identify novel ER-resident proteinsnvolved in Ca2+ signaling, we obtained a cDNA fragmentncoding a putative signal peptide and the following partialrotein from a mouse brain library. Subsequent full-lengthloning defined the primary structure of a novel protein com-osed of 483 amino acid residues. Homology searches usinghe BLAST program showed that this protein is conserved

cross animal species (Fig. S1), and the murine protein has3% overall identity to the C. elegans counterpart. However,here is no structural homologue in the yeast genomes. Thisrotein, namely calumin, consists of an amino-terminal signal

86 M. Zhang et al. / Cell Calcium 42 (2007) 83–90

Fig. 1. Identification of calumin. (A) Structural features of calumin. Proposed domain structures of calumin are schematically illustrated. SP, signal peptide; lum,ER-luminal domain; TM, transmembrane segment; cyt, cytoplasmic domain; cc, coiled-coil. (B) Northern blot analysis of calumin in mouse tissues. Total RNApreparations (10 �g in each tissue) were analyzed using the hybridization probe derived from calumin cDNA. (C) Western blot analysis of calumin in mousetissues. Total microsomal proteins (10 �g/lane) prepared from adult mice were analyzed with mAb29. (D) Subcellular localization of calumin in the mousecerebellum. Fractionated preparations (5 �g protein/lane) were subjected to immunoblotting with specific antibodies indicated. (E) Indirect immunofluorescenceanalysis of calumin. HEK293 cells expressing C-terminally tagged calumin (calumin-FLAG) together with either pDsRed2-ER or pDsRed2-Mito were analyzedu min carc trations

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sing a confocal microscope. Scale bar, 5 �m. (F) Topology analysis of caluells expressing FLAG-tagged calumin were treated with indicated concenubjected to immunoblot analysis using antibodies to FLAG or PDI.

eptide, a putative ER-luminal domain, a transmembrane seg-ent, and a carboxyl-terminal cytoplasmic domain (Fig. 1A).database survey did not find any consensus motifs in the

rimary structure of calumin other than a putative coiled-coilegion at its carboxyl-terminal end. Northern blot analysisetected a 3.5-kb calumin mRNA in all of mouse tissuesxamined (Fig. 1B).

In immunoblot analysis, mAb raised against the cyto-lasmic portion specifically recognized a protein with anpparent molecular mass of ∼60 kDa recovered in micro-omal preparations from various tissues (Figs. 1C and S2).ubcellular distribution was further analyzed with fraction-ted samples prepared from the mouse cerebellum and theractionation quality was confirmed by enrichment of ref-rence proteins into the respective fractions: CNX, an ERarker; G�q/11, a plasma membrane marker; GM130, aolgi marker; COX4, a mitochondria marker (Fig. 1D). Theistribution profiles of calumin and CNX were indistinguish-

ble. When the expression plasmid carrying FLAG-taggedalumin cDNA was introduced into cultured cells, the recom-inant protein colocalized with the ER marker DsRed-ERFig. 1E), but not with the mitochondrial marker DsRed-

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rying FLAG tag at its N- or C-terminus. Membrane vesicles prepared froms of trypsin (�g/ml) in the absence or presence of 1% Triton X-100, and

ito, further supporting the ER residence of calumin. Tonvestigate the membrane topology of calumin, the FLAGag was introduced into either the C-teminus of calumincalumin-FLAG) or the N-terminal region preceded by theignal peptide (FLAG-calumin). Microsomal vesicles wererepared from transfected cells under isotonic conditions andubjected to proteolytic digestion using the intraluminal pro-ein PDI as an internal control. Calumin-FLAG progressivelyisappeared with increasing concentrations of trypsin, whileleaved fragments retaining the tag sequence were detectedn the case of FLAG-calumin (Fig. 1F). The observationsuggest that calumin is an ER-resident membrane-spanningrotein in various cell types and that its N- and C-terminusre faced to the lumen and the cytoplasm, respectively.

.2. Ca2+-binding activity of calumin

Of 116 amino acid residues in the ER-luminal domain of

alumin, 46 are acidic residues. This observation led us tonvisage that calumin could bind Ca2+. To test this possi-ility, GST fusion proteins carrying the luminal (GST-lum)nd cytoplasmic (GST-cyt) domains were produced in bac-

M. Zhang et al. / Cell Calcium 42 (2007) 83–90 87

Fig. 2. Ca2+-binding activity of calumin. (A) Production of GST fusion pro-teins carrying the luminal domain of calumin (GST-lum) and the cytoplasmicdomain (GST-cyt). (B) Stain All-staining of GST-calumin fusion proteins.Total bacterial extracts (30 �g/lane) were examined by Coomassie brilliantblue staining (CBB) or Stains All staining. (C) 45Ca2+ overlay assay of fusionprotein carrying the ER-luminal domain. Purified fusion proteins (5 �g/lane)were separated on SDS-polyacrylamide gels, blotted onto nylon membranes,and subjected to CBB staining (left panel) and 45Ca2+ overlay assay (autora-dtc

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Fig. 3. Impaired Ca2+ signaling in calumin-knockout MEFs. (A) Weak Ca2+

responses of calumin-knockout MEFs to CPA. Fura 2-measurements wereperformed with MEFs derived from six embryos of each genotype (∼50cells per embryo), and normalized traces are shown. (B) Normal restingCa2+ levels of calumin-knockout MEFs in 2 mM Ca2+ and nominally Ca2+-free bathing solutions. (C) Reduced CPA-induced Ca2+ release in calumin-knockout MEFs. (D) Weak SOCE in calumin-knockout MEFs. *P < 0.05 inSok

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iogram in right panel). Data in (B and C) are representative of at leasthree independent experiments. (D) 45Ca2+ microdialysis of fusion proteinarrying the ER-luminal domain. Data represent mean ± S.E.M. (n = 6).

eria (Fig. 2A), and subjected to staining with Stains All, aye generally used to visualize Ca2+-binding proteins. Theye reacted with GST-lum but not with GST-cyt, suggestinga2+-binding activity for the luminal domain (Fig. 2B). Ca2+-inding to GST-lum was further confirmed by the 45Ca2+

verlay assay (Fig. 2C). Moreover, the 45Ca2+ microflowialysis demonstrated that the luminal domain binds 15 molf Ca2+/mol of protein with an apparent Kd of ∼0.75 mMFig. 2D). Therefore, calumin appears to be a Ca2+-bindingrotein with a high capacity and a moderate affinity.

.3. Contribution of calumin to Ca2+ handling

To examine the physiological role of calumin, knockoutice were established by our standard gene-targeting meth-

ds. Neither calumin mRNA nor protein was detected inhe homozygous mutant mice generated, indicating that thentroduced mutation is a null mutation of the gene (Fig. S2).

ore than half of calumin-knockout mice were decimated at

arly embryonic stages, and the rest survived during embry-nic development but died shortly after birth (unpublishedbservation). Although the direct cause of death in eachevelopmental stage remains to be investigated, the lethal-

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tudent’s t-test. Values represent mean ± S.E.M. (E) Immunoblot analysisf major Ca2+ store-related proteins in MEFs. WT, wild-type; KO, caluminnockout.

ty suggests that calumin plays indispensable physiologicaloles in certain cell types.

Because calumin seems to be an ER Ca2+-binding pro-ein ubiquitously expressed, we examined whether caluminas vital for cellular Ca2+ homeostasis in MEFs from thenockout mice. Calumin-knockout MEFs were morphologi-ally normal, and retained cell growth similar to the wild-typeells, as determined by flow cytometric measurement of car-oxyfluorescein succinimidyl ester dye dilution (data nothown). Fura 2-Ca2+ imaging detected no abnormality in the

esting Ca2+ level of calumin-knockout MEFs in normal anda2+-free bathing solutions (Fig. 3A and B). In contrast, theutant MEFs showed a 20–30% reduction in the amplitude of

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Fig. 4. Enhanced vulnerability of calumin-knockout MEFs to ER stress.MEFs were cultured for 24 h with (A), TG; (B), TM; (C), STS; or (D),TNF-� plus cycloheximide (10 �g/ml) at indicated concentrations. Percent-ages of cells positive for annexin V were flow cytometrically determinedin triplicates. Data represent mean ± S.E.M. Statistical differences betweencia

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a2+ transients evoked by CPA, a SERCA inhibitor (Fig. 3And C). Ca2+ transients triggered by either TG (Fig. S3B)r ionomycin (data not shown) were also impaired in theutant MEFs. Meanwhile, the mutant MEFs retained nor-al expression of major Ca2+-handling proteins includingERCA, CNX and CRT (Fig. 3E). Therefore, ER Ca2+ con-

ents were significantly decreased in the mutant MEFs likelyue to the loss of calumin.

Ca2+ depletion of intracellular stores triggers SOCE, aey-signaling pathway for cellular Ca2+ homeostasis sharedy various cell types [9]. Upon application of extracellulara2+ after store depletion induced by CPA, MEFs dis-layed clear SOCE responses. However, the average peakmplitude of SOCE in the mutant MEFs was reduced by0–30% compared with that of wild-type cells (Fig. 3And D). Therefore, SOCE activity is partially impaired inalumin-knockout MEFs.

To verify poor Ca2+ signaling in calumin-knockout MEFs,e next designed a rescue experiment using retrovirus-ediated expression of recombinant calumin carrying theLAG tag. The reintroduction of full-length calumin into theutant MEFs restored both TG-elicited Ca2+ transients andOCE to levels similar to those observed in mock-infectedild-type MEFs (Fig. S3). In contrast, such restoration wasot achieved by the calumin mutant lacking the cytoplasmicortion. It is thus suggested that calumin deficiency is the pri-ary cause of insufficient Ca2+ signaling in the mutant MEFs

nd that the structural integrity of calumin is a prerequisiteor its physiological function.

.4. Calumin and stress-induced cell death

Ca2+ depletion in the ER reservoir results in the stressesponse termed the UPR, and the disorganized interplayetween Ca2+ signals and stress signals seems to induce celleath [20]. To survey the potential role of calumin in UPR-ssociated cell death, the effects of ER stressors, such as TGnd TM, were examined in calumin-knockout MEFs. Exter-alization of phosphatidylserine on the plasma membrane isssociated with early apoptosis and can be detected by cell-urface staining with annexin V, a calcium-dependent phos-holipid binding protein [21]. Flow cytometric quantificationf annexin V-positive cells revealed that calumin-knockoutEFs are more sensitive to both TG- and TM-induced cell

eath than wild-type controls (Fig. 4). On the other hand, bothroups of MEFs were killed to a similar extent upon treat-ent with STS as a broad-spectrum kinase inhibitor known

o induce apoptosis via the mitochondrial pathway [22] orith TNF-� as a direct inducer of caspase-mediated apoptosis

23]. The observations clearly suggest that calumin specifi-ally takes part in stress responses originating from the ER.

.5. Calumin and UPR signaling

In higher eukaryotes, three distinct UPR signaling path-ays are stimulated upon ER stress, which are mediated

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alumin-knockout (KO) and wild-type (WT) MEFs are marked with aster-sks (*P < 0.05 and **P < 0.01 in Student’s t-test). Representative data fromt least three independent experiments are shown.

y sensors/transducers including inositol-requiring trans-embrane kinase and endonuclease 1� (IRE1�), protein

inase-like ER kinase (PERK), and activation of transcrip-ion factor 6 (ATF6) [24]. ER stress initiates IRE1�-mediatenconventional splicing of the xbp1 transcript, the abundancef which is increased by the IRE1�-ATF6 collaboration.roduced XBP1, in turn, causes transcription of severalenes involved in ER-associated protein degradation. IRE1�urthermore activates JNK via binding to tumor necrosisactor-associated factor 2 (TRAF2). PERK, another upstreamediator, transiently shuts off protein translation by phos-

horylation of eIF2�. These molecular events took placeomparably between wild-type and calumin-knockout MEFsFig. S4). In the support of successful UPR initiation, normalnduction of downstream targets such as BiP and CHOP wasbserved in the mutant MEFs (Fig. 5A).

Although it is still unclear why the UPR initially pro-ects cells from the ER stress and eventually triggers celleath, physiological functions of caspase family members areequired for these processes [23]. Therefore, the activation ofhe apical caspases, caspase-9 and -12, or of the executioneraspase, caspase-3, was assessed by their cleavage from pro-

aspases to corresponding mature forms. Caspase pathwaysere found to be not significantly affected in the absencef calumin (Fig. 5B). Therefore, calumin-knockout MEFseem to retain normal pathways for UPR and caspase activa-

M. Zhang et al. / Cell Calciu

Fig. 5. Apparent normal caspase activation in TG-treated calumin-knockoutMEFs. MEFs were treated with TG (1 �M) for the times indicated andlysed for the following analyses. (A) Detection of BiP and CHOP inductionby immunoblotting. Actin was blotted as a loading control. (B) Detectionof cleaved caspases by immunoblotting. Representative data from at leasttd

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hree independent experiments are shown and no significant differences wereetected between calumin-knockout (KO) and wild-type (WT) MEFs.

ion. The observations suggest that calumin could operate ons-yet-unidentified signaling pathways protective against celleath, dysregulation of which might account for the increasedulnerability of calumin-knockout MEFs to pharmacologi-ally elicited ER stress.

. Discussion

Our present data clearly suggest that calumin plays anmportant role in the ER Ca2+ storage function by bindinga2+ via its ER-luminal portion. Free Ca2+ concentrations

n the ER are estimated to be 0.3∼0.8 mM [25]. In theresence of physiological concentrations of KCl, the lumi-al domain of calumin binds Ca2+ with a moderate affinityKd = ∼0.75 mM), which is close to the free calcium level inhe ER of resting cells. In light of its Ca2+-binding properties,alumin may be an ideal candidate for an ER Ca2+ sensornd/or signal transducer. Of several Ca2+-binding proteinsithin internal stores, junctate and STIM1 were implicated

n the cellular machinery for SOCE [6–8]. It may be possiblehat calumin, together with these proteins, controls SOCE inarious cell types, because calumin-knockout MEFs showediminished SOCE (Fig. 3).

b2

m 42 (2007) 83–90 89

Calumin-knockout MEFs exhibited enhanced cell deathy ER stress-inducing agents (Fig. 4). On the other hand,here was no significant difference in death-associated read-uts in known UPR pathways between calumin-knockout andild-type MEFs (Figs. 5 and S4). Therefore, the decreased

tress resistance observed in the mutant MEFs may implyefective pro-survival signals rather than augmented anti-urvival signals. Considering that Ca2+ influx across thelasma membrane contributes to cell fate decision [26],educed SOCE might underlie proposed signaling defects inhe mutant MEFs. For example, moderate Ca2+ influx in neu-onal cells elicits Ca2+-dependent signaling pathways, whichonverge on the transcription factor cAMP-response elementinding (CREB) protein to induce pro-survival genes [27].uclear factor of activated T cells (NFAT) is likely activatedy Ca2+ entry via SOCE in MEFs [28] and NFAT-inducedene expression seems to be essential for a wide variety ofevelopmental processes [29]. In calumin-knockout MEFs,oor NFAT activation due to attenuated SOCE could leado enhanced cell death under the stress conditions. Althoughecreased ER Ca2+ contents could protect cells against ER-tress-induced death according to previous findings [30–33],ur observations may suggest that this mechanism did notffectively function in calumin-knockout MEFs. It might behat the disadvantage conferred by poor SOCE activity over-ides the potential advantage granted from low Ca2+ storage,esulting in a net outcome of facilitated cell death in calumin-nockout MEFs.

The present findings suggest that calumin, as an ER Ca2+-inding protein, is involved in cellular signaling for SOCEnd ER stress. The cytoplasmic domain could be implicatedn the proposed signaling pathway by protein–protein inter-ctions and, thus, it is important to identify predicted bindingartners in future studies. Exploration of modes of action foralumin should provide not only a cue to elucidate the molec-lar basis of the calumin-knockout mice abnormalities, butlso a novel paradigm for ER-emanated signaling pathways.

cknowledgements

We thank Ms. Miyuki Kameyama, Ms. Nozomi Sasakind Ms. Sae Aoki for technical assistance in mutant mouseeneration and flow cytometric analysis. This work was sup-orted in part by grants from the Ministry of Education,ulture, Sports, Science and Technology of Japan, the Min-

stry of Health and Welfare of Japan, the Naito Foundation,he Sumitomo Foundation, the Uehara Memorial Foundation,he Takeda Science Foundation, and the 21st Century Centerf Excellence program.

ppendix A. Supplementary data

Supplementary data associated with this article cane found, in the online version, at doi:10.1016/j.ceca.006.11.009.

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