Loss of the Atp2c1 Secretory Pathway Ca2�-ATPase (SPCA1)in Mice Causes Golgi Stress, Apoptosis, and MidgestationalDeath in Homozygous Embryos and Squamous Cell Tumorsin Adult Heterozygotes*

Received for publication, April 10, 2007, and in revised form, June 13, 2007 Published, JBC Papers in Press, June 27, 2007, DOI 10.1074/jbc.M703029200

Gbolahan W. Okunade‡, Marian L. Miller§, Mohamad Azhar‡, Anastasia Andringa§, L. Philip Sanford‡,Thomas Doetschman‡, Vikram Prasad‡, and Gary E. Shull‡1

From the Departments of ‡Molecular Genetics, Biochemistry, and Microbiology and §Environmental Health,University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524

Loss of one copy of the human ATP2C1 gene, encoding SPCA1(secretory pathwayCa2�-ATPase isoform1), causesHailey-Haileydisease, a skindisorder.Weperformed targetedmutagenesisof theAtp2c1 gene in mice to analyze the functions of this Golgi mem-brane Ca2� pump. Breeding of heterozygous mutants yielded anormalMendelianratioamongembryosongestationday9.5;how-ever, null mutant (Spca1�/�) embryos exhibited growth retarda-tion and did not survive beyond gestation day 10.5. Spca1�/�

embryos had an open rostral neural tube, but hematopoiesis andcardiovascular development were ostensibly normal. Golgi mem-branes of Spca1�/� embryos were dilated, had fewer stacked leaf-lets, andwereexpandedinamount,consistentwith increasedGolgibiogenesis. The number of Golgi-associated vesicles was alsoincreased, and roughendoplasmic reticulumhad fewer ribosomes.Coated pits, junctional complexes, desmosomes, and basementmembranes appeared normal in mutant embryos, indicating thatprocessing and traffickingof proteins in the secretory pathwaywasnot massively impaired. However, apoptosis was increased, possi-bly the result of secretory pathway stress, and a large increase incytoplasmic lipidwasobserved inmutant embryos, consistentwithimpaired handling of lipid by the Golgi. Adult heterozygous miceappeared normal and exhibited no evidence of Hailey-Hailey dis-ease; however, aged heterozygotes had an increased incidence ofsquamous cell tumors of keratinized epithelial cells of the skin andesophagus. These data show that loss of the Golgi Ca2� pumpcauses Golgi stress, expansion of the Golgi, increased apoptosis,andembryonic lethality anddemonstrates thatSPCA1haploinsuf-ficiency causes a genetic predisposition to cancer.

The mammalian Ca2�-transporting ATPases include twosecretory pathway Ca2�-ATPases (SPCAs)2 (1–3), three sarco-

(endo)plasmic reticulum Ca2�-ATPases (4), and four plasmamembrane Ca2�-ATPases (5, 6). The biochemical, cell biolog-ical, and physiological functions of SERCA and plasma mem-brane Ca2�-ATPases have been intensively studied (4, 7). Onlylimited information is available for the SPCAs (8), which areclosely related to PMR1, a P-type Ca2�-ATPase in yeast that isexpressed in Golgi membranes (9) and transports both Ca2�

andMn2� (10). Loss of PMR1 affects outer chain glycosylation,proteolytic processing, and trafficking of proteins in the secre-tory pathway (9). Inmammals, SPCA1 is expressed in all tissues(1), whereas SPCA2 is expressed in only a limited set of tissues(3). Like PMR1, both SPCA1 and SPCA2 are localized to theGolgi and transport Ca2� and Mn2� (3, 11). There is evidencethat the cell biological functions of SPCA1 are also similar tothose of PMR1 (12).Loss of one copy of the human ATP2C1 gene, encoding

SPCA1, causes Hailey-Hailey disease (HHD), an autosomaldominant skin disorder (13, 14). SPCA1 protein levels in HHDkeratinocytes are reduced to about half of normal levels, andGolgi Ca2� handling is impaired (15). HHD is similar to Darierdisease, which is caused by null mutations in one copy of thehuman ATP2A2 gene, encoding SERCA2 (16). Both diseasesare characterized by acantholysis (a disruption of cell-cell con-tacts) in the suprabasal layers of the skin. As themajor ER Ca2�

pump in most tissues, including keratinocytes, the function ofSERCA2 is similar to that of SPCA1 in that it maintains luminalCa2� concentrations in a major compartment of the secretorypathway. In mice, SERCA2 haploinsufficiency does not causeDarier disease but does lead to squamous cell tumors of kera-tinized epithelial cells (17, 18), the same cell type affected inDarier disease. In humans, a low incidence of squamous celltumors has been reported in both Darier disease (19) and HHD(20, 21), but it is unclear whether this is a chance association oris caused by the reduction in Ca2� pump levels and activity.In the current study, we developed a gene-targeted mouse

model for SPCA1 and analyzed the phenotype resulting fromheterozygous and homozygous null mutations. The resultsshow that SPCA1 null embryos undergo a substantial degree of

* This work was supported by National Institutes of Health Grants HL61974,DK50594, and ES06096. The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

1 To whom correspondence should be addressed: Dept. of Molecular Genet-ics, Biochemistry, and Microbiology, University of Cincinnati College ofMedicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0524. Tel.: 513-558-0056; Fax: 513-558-1885; E-mail: shullge@ucmail.uc.edu.

2 The abbreviations used are: SPCA, secretory pathway Ca2�-ATPase; SERCA,sarco(endo)plasmic reticulum Ca2�-ATPase (the number following SPCAor SERCA refers to the specific isoform); HHD, Hailey-Hailey disease;

Spca1�/�, Spca1�/�, and Spca1�/�, SPCA1 wild-type, heterozygous, andhomozygous mutant mice, respectively; ED, embryonic day; ER, endoplas-mic reticulum; RER, rough ER; H&E, hematoxylin and eosin; UPR, unfoldedprotein response.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 36, pp. 26517–26527, September 7, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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structural development and survive until gestation day 10.5.However, embryonic tissues exhibited a high incidence of apo-ptosis and ultrastructural evidence of severe Golgi stress, thusestablishing SPCA1 deficiency as the first clear example of acondition that causes Golgi stress. Heterozygous mutantsexhibited no evidence of HHD but did develop squamous celltumors, as observed in SERCA2 heterozygous mice. The paral-lels between the effects of SPCA1 deficiency and SERCA2 defi-ciency are consistent with a model in which species differencesin the balance between prosurvival and proapoptotic responsesof keratinocytes to secretory pathway stress favor developmentof cancer in mice and acantholytic skin disease in humans.

EXPERIMENTAL PROCEDURES

Generation of Mutant Mice—A 2.1-kb BamHI-HindIII frag-ment beginning in intron 7 and ending in intron 9 and a 2.4-kbBamHI-PstI fragment beginning in intron 13 and extending tointron 15were inserted into the pMJKO vector (22). The vectorcontained the neomycin resistance and HSV-thymidine kinasegenes for positive and negative selection. Electroporation ofembryonic stem cells and blastocyst-mediated transgenesiswere performed as described previously (22), and male chi-meric mice were bred with Black Swiss females. Southern blotanalysis was performed using a 3� outside probe correspondingto a 1.25-kb PstI-BamHI site from intron 15 and 5� inside probecorresponding to codons 182–228 from exon 8. The mice usedin the aging study were from early generations and of the orig-inal mixed 129/SvJ and Black Swiss background; mice used foranalysis of the embryo lethality phenotype had been back-crossed onto the Black Swiss background. The morning onwhich a vaginal plug was observed was regarded as embryonicday (ED) 0.5.Genotype Analysis—Genotyping was performed by Southern

blot analysis of tail or spleen DNA or by PCR analysis of tailDNA,whole embryos, or embryonic yolk sac using a set of threeprimers. A 276-bp product from the wild-type allele was ampli-fied using forward (5�-GCTCGCATGCAAATGTTGCATCC-TGTA-3�) and reverse (5�-ACTGAGAACTATGCATCCCA-CTGAG-3�) primers corresponding to nucleotides 475–501and 725–750 of intron 9, respectively. This fragment containedthe HindIII site used to prepare the 5� arm. A 188-bp productfrom themutant allele was amplified in the same reaction usingthe forward primer described above and a reverse primer (5�-GCATGCTCCAGACTGCCTTG-3�) based on a sequence inthe promoter of the neomycin resistance gene.Northern Blot and PCR Analysis of Mutant and Wild-type

SPCA1 mRNAs—Total RNA was isolated from tissues of adultwild-type andheterozygousmice and fromembryos at differentstages of development. Northern blot analysis of RNA fromadult tissues was performed as described previously (22) usingan SPCA1 cDNA probe and an L32 ribosomal protein cDNAprobe as a loading control. For PCR analysis, first strand cDNAwas prepared using oligo(dT) primers and reverse tran-scriptase. For adult tissues, PCR primers corresponded tocodons 182–190 in exon 8 and the reverse complement ofcodons 428–435 in exon 15. For embryos, PCR primers corre-sponded to codons 182–190 and the reverse complements ofeither codons 366–373 in exon 13 or codons 660–667 in exon

21. PCR products were analyzed by agarose gel electrophoresisand DNA sequencing.Immunoblot Analysis of SPCA1Protein—Newborn pups (day

1) were euthanized, and back-skins were collected and incu-bated in 1� Dispase II (Roche Applied Science) overnight at4 °C with gentle agitation. After incubation, the epithelial layer,consisting of keratinocytes, was peeled off the dermal layer,frozen in liquid nitrogen, and stored at�80 °C until further use.Epithelial samples were manually homogenized as describedbefore (17). Adult mouse hearts were manually pulverized inliquid nitrogen and homogenized in bufferH (10mMTris, 1mMEDTA, 0.25% Nonidet P-40, 2 mM dithiothreitol, containing amixture of protease and phosphatase inhibitors) using a Poly-tron 3000 mechanical homogenizer. Protein concentrationswere determined using the Coomassie protein assay reagent(Pierce). Proteins were resolved on a discontinuous, reducing9% SDS-polyacrylamide gel, transferred to nitrocellulosemem-branes, and probed using an anti-SPCA1 antibody (23) andhorseradish peroxidase-conjugated anti-rabbit secondary anti-bodies (KPL Inc., Gaithersburg, MD). Chemiluminescence wasdeveloped using the LumiGlo chemiluminescent substrate kit(KPL), and autoradiograms were developed using BioMaxFilms ML and MR (Eastman Kodak Co.).Tissue Preparation—Tissueswere fixed for light and electron

microscopy and morphometry, as described previously (22).Histology and LightMicroscopicMorphometry—Paraffin sec-

tions of embryos were stained with H&E (93 pups). Placentawas analyzed, and morphometry of yolk sac vessels was per-formed using H&E-stained sections of embryos in utero. At�40 magnification, the area (�m2) of vessel lumen (bloodislands) was determined, and the number of visceral endoder-mal yolk sac and mesenchymal cell nuclei/�m of yolk sac wasdetermined. In addition, the mitotic index and the number ofvisceral yolk sac cells containing more than three lipid dropletswas counted. The thickness of visceral yolk sac endoderm wasdigitized (�m), as was the nuclear area (�m2).Plastic blocks containing embryos were sectioned at 2 �m

thick and stained with toluidine blue for light microscopy andlight microscopic morphometry. Other embryo blocks werefirst trimmed to the level of the heart, where 2-�m-thick sec-tions weremade. All null embryos were grossly smaller and lesswell developed than age-matchedwild-type embryos at ED9.5–10.5. Tissue sites were identified according to an atlas of mousedevelopment (24). Data were obtained from anatomical sites asfollows: dorsal and ventral rostral neural tube, dorsal and ven-tral headmesenchyme, dorsal and ventral distal neural tube andmesenchyme, limb budmesenchyme, surface ectoderm, bulbuscordis and outflow tract, branchial archmesenchyme, primitivegut, otic and optic placodes, blood vessels, and red blood cells.Within these sites, the percentage of each of the following wasdetermined: 1) mitosis (late prophase through late telophase),2) apoptotic cells and apoptotic debris, and 3) cells containingmore than three fat droplets.Electron Microscopic Morphometry—The cell ultrastructure

of eight Spca1�/� and 11 Spca1�/� embryos was examinedfrom selected sites (rostral neural tube, heart, and yolk sac).Thin sections were placed on naked copper grids, stained withuranyl acetate and lead citrate, and photographed atmagnifica-

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tions ranging from�2500 to�14,000. The number of embryosused for morphometry was as follows: ED 8.5, four Spca1�/�,two Spca1�/�; ED 9.5, four Spca1�/�, three Spca1�/�; ED 10.5,three Spca1�/�, three Spca1�/�. The number of cells analyzedby electron microscopy was just over 1000. Micrographs wereread blind to both genotype and age. An initial semiquantitativeassessment (1� to 5�) of the number of ER profiles per cellprofile, the degree of dilation of the Golgi, and the extent towhich ribosomes were lost from the RER was made, and thenmorphometrywas performed. The incidence, per cell profile, ofmitochondria, desmosomes, coated pits, and junctional com-plexes was determined. In addition, the Vd (volume densities)of RER, Golgi, Golgi-associated ribosome-free vesicles, andtotal ER was determined from negatives of �10,000 or�14,000, using a grid applied to each print. Ribosomes permeasured length of RER and Golgi-associated dense vesicleswere counted.Statistical Analyses—Excel was used for managing the data,

and PROC GLM and PROC MIXED (SAS 9.1; Cary, NC) andpaired and unpaired t tests (SigmaPlot, SYSTAT, Point Rich-mond, CA) were used for analyses. Results were reported asmeans � S.E. of the mean. p � 0.05 was considered significant.

RESULTS

Generation of Mice with a Null Mutation in the Spca1Gene—Embryonic stem cells carrying a null mutation in theSpca1 gene were prepared using a construct in which exons

10–13 were replaced with the neomycin resistance gene (Fig.1A). Because these exons encode both the catalytic phospho-rylation site and transmembrane domains 3 and 4, their exci-sion ensured that themutationwould be a functional null. Blas-tocyst-mediated transgenesis yielded chimeric male mice thattransmitted the mutant allele through the germ line (Fig. 1B).PCR genotyping of tail DNA of live offspring (Fig. 1C) of het-erozygous breeding pairs revealed that only wild-type and het-erozygous pups, in a 1:2 ratio (228:425), were present at birth,indicating that the homozygous null mutation caused embry-onic lethality. Spca1�/� mice exhibited no evidence of skinlesions indicative of Hailey-Hailey disease, and there were nohistologic abnormalities, except in aged mice, where there wasan increased incidence of squamous cell tumors (see below).Northern blot analysis of RNA from Spca1�/� and wild-

type tissues identified SPCA1 mRNAs of �4.6–4.8 kb in alltissues examined (Fig. 1D). For some tissues, such as stom-ach, liver, and colon, heterozygotes exhibited a reduction insignal intensity, consistent with loss of the mutant mRNA,whereas in others there was a broadening of the mRNA sizein Spca1�/� samples, consistent with the expression of bothwild-type and mutant mRNAs. PCR and DNA sequenceanalysis confirmed that Spca1�/� tissues contained amutant mRNA in which the donor site of exon 9 was splicedto the acceptor site of exon 14, thereby producing an mRNAthat lacked codons 252–373 (Fig. 1E).

FIGURE 1. SPCA1 gene targeting, genotyping, and mRNA analysis. A, targeting strategy. Top, structure of the wild-type allele. Middle, targeting construct,with the neomycin resistance gene (Neo) replacing sequences from exons 10 –13 and the herpes simplex virus thymidine kinase gene (TK) included for negativeselection. Bottom, structure of the targeted allele. The locations of the wild-type 9.7- and 5-kb fragments and the mutant 8.5-kb fragment are shown above eachallele, and the diagnostic probes are indicated above the wild type allele. B, Southern blot analysis of wild-type (�/�) and targeted (�/�) embryonic stem (ES)cell DNA and spleen DNA of wild-type and heterozygous mice using the indicated enzymes and probes. C, PCR genotyping revealed wild-type and heterozy-gous offspring but no homozygous mutant offspring. D, RNA (20 �g/lane) from the indicated tissues (Stom, stomach; Mamm, mammary gland) was hybridizedwith cDNA probes for SPCA1 and the L32 ribosomal subunit (as a loading control). E, reverse transcription (RT)-PCR analysis of SPCA1 mRNA from Spca1�/� andSpca1�/� mice using primers from exons 8 and 15 and agarose gel electrophoresis. DNA sequence analysis revealed a 366-bp difference in size between thewild-type (WT) and knock-out (KO) PCR products, which corresponded to a deletion of exons 10 –13.

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Embryonic Lethality of Null Mutants Occurs at GestationDay 10.5—Heterozygous mice were mated, and embryos wereexamined at various stages of gestation. PCR genotypingrevealed the presence of ostensibly live null mutants as late asED 10.5 (Fig. 2A), and reverse transcriptase-PCR analysis ofRNA from null embryos at day 8.5 confirmed the absence ofwild-type SPCA1 mRNA and the presence of mutant SPCA1mRNA (Fig. 2B). On EDs 7.5 and 8.5, there was no evidence ofincreased death among null mutants (12 �/�, 38 �/�, and22�/�). A normalMendelian ratio for Spca1�/� embryos wasalso observed on ED 9.5 (56 �/�, 116 �/�, and 54 �/�). OnED 10.5, however, the number of null mutants was reduced (38�/�, 86 �/�, and 20 �/�), and by ED 11.5 and later, live nullmutants were not observed.Reduction in SPCA1Protein Levels inHeterozygousTissues—As

discussed below, keratinocytes are the only cell type or tissueexhibiting a clear phenotype in adult tissues, and the heart exhib-ited no apparent structural or functional abnormalities even inhomozygous null embryos. Immunoblot analysis of both the epi-thelial (keratinocyte) layer of newborn mouse skin (Fig. 3A) andadult heart (Fig. 3B) showed that the loss of one allele caused asignificant reduction in SPCA1 protein levels in both tissues.Incomplete Closure of Neural Tube in Null Mutant Em-

bryos—The most striking gross defect in Spca1�/� embryoswas failure of neural tube closure (Fig. 4). During the early

stages of neural tube development on ED8.5, nullmutants weremorphologically indistinguishable from Spca1�/� andSpca1�/� embryos, and there was no apparent growth retarda-tion. Primary neurulation appeared to have proceeded nor-mally in Spca1�/� embryos, with development of the notocord,neural plate, neural crests, and epidermis; the caudal and ven-tral aspects of the neural tubewere similar among all genotypes,and successful closure of the caudal portion of the neural tube

FIGURE 2. PCR analysis of genomic DNA and SPCA1 mRNA during embry-ogenesis. Timed matings of heterozygous breeding pairs were performed,and DNA or RNA was isolated from embryos collected at various times duringgestation. PCR products were separated by agarose gel electrophoresis.A, PCR analysis of genomic DNA revealed the presence of all three genotypeson ED 8.5, 9.5, and 10.5 (wild type, 276 bp; mutant, 188 bp). B, reverse tran-scriptase-PCR analysis of SPCA1 mRNA from ED 8.5 embryos using primer setsthat amplify only a 575-bp fragment for the wild-type allele (four samples onleft) or fragments for both the wild-type (1454-bp) and null (1088-bp) alleles(four samples on right). Wild type, �/�; heterozygous, �/�; null mutant,�/�. M, molecular weight markers.

FIGURE 3. Reduced SPCA1 expression in epithelial and cardiac tissues ofSpca1�/� mice. Immunoblot analysis of whole protein preparations fromback-skin epithelia (A) and heart (B) was performed using an anti-SPCA1 anti-body, with tubulin and sarcomeric (s.) actin serving as loading controls. Den-sitometric analysis of the autoradiograms revealed that SPCA1 protein levelsin skin epithelia and heart were 55.9 � 0.5 and 56.3 � 0.6% of wild-type levels,respectively (p � 0.05).

FIGURE 4. Failure of rostral neural tube closure in Spca1�/� embryos.A, Spca1�/� and Spca1�/� embryos from the same litter at ED 10.5. The neuraltube (white arrows) is closed in the wild type but open in the null mutant. Notethe appearance of the neuroectoderm in the area of the rhombencephalon,which is ruffled in the mutant (red arrow). B, H&E-stained sections of wild-typeand null mutant embryos at ED 9.5, fixed in utero. The neural tube is closed inthe wild type and open in the mutant, which also exhibits ruffling of theneuroectoderm (red arrow). N, neuroectoderm; M, mesenchyme. Bar, 100 �m.

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occurred by ED 8.5. By ED 9.5, however, Spca1�/� embryosexhibited a failure of neural tube closure from the hind brainrostrally. Around ED 9.5, the presumptive nerve cells in therostral neural tube had developed neural processes containingneurotubules. Subjectively, these appeared to be less well devel-oped in mutants than in wild-type embryos, but they occurredat the appropriate interface between the mesenchyme and theopen rostral neural tube. Spca1�/� embryos that survived toED 10.5 were growth-retarded and continued to have an openrostral neural tube, and ruffling of the edges of the open neuraltube was observed in some of the mutants (red arrows in Fig. 4,A and B). The medial hinge point of the neural tube oftenappeared appropriate in Spca1�/� embryos, but dorsolateralhinging was incomplete from the hind brain forward. Despitegrowth retardation and nonclosure of the rostral neural tube innull embryos, some growth and development continuedbetween EDs 8.5 and 10.5, and, very infrequently in olderSpca1�/� embryos, partial closure of the neural tube occurredat the maxilla and face.Hematopoiesis and Cardiovascular Development Appear

Normal in Null Mutant Embryos—Despite their dramaticappearance, neural tube defects cannot account for the observedembryonic death at ED 10.5. However, because impaired hemato-poiesis or cardiovascular development can be a major cause ofdeath during this period, we examined these processes.

On ED 9.5, yolk sac blood islands (Fig. 5A), red blood cells(Fig. 5B), and blood vessels, including the dorsal aorta (Fig. 5C)and yolk sac vessels, appeared to be similar in Spca1�/� andSpca1�/� embryos, indicating that hematopoiesis and vasculardevelopment were relatively normal at this stage. Specific indi-cators of yolk sac health were measured histologically at ED 9.5in an attempt to identify and quantify any subtle impairments ofdevelopment thatmight be a forerunner of embryonic death onED 10.5. Themitotic index and percentage of yolk sac cells withmore than three lipid droplets (see below)were not significantlydifferent between Spca1�/� and Spca1�/� embryos, and apo-ptosis was not elevated. Visceral endodermal thickness wassimilar in Spca1�/� and Spca1�/� embryos (21.8 � 3.1 and19.31 � 1.94 �m, respectively), and the numbers of both vis-ceral endodermal cells/�m (0.11 � 0.02 in Spca1�/�; 0.09 �0.01 in Spca1�/�) and mesodermal cells/�m (0.05 � 0.01 inSpca1�/�; 0.04 � 0.01 in Spca1�/�) were essentially the same.Finally, there was no significant difference between the twogenotypes in the nuclear area of visceral endodermal cells(52.3 � 3.15 �m2 in Spca1�/�; 54.6 � 3.06 �m2 in Spca1�/�),as would be expected if yolk sac cells were blocked in either Sphase or G2 or exhibited severe aneuploidy.

Cardiac function and development appeared to be grosslynormal in null mutants. The hearts of ED 9.5 and 10.5 embryosstopped beating when the embryos were removed from theuterus; however, spontaneous beating could be elicited by gen-tlemechanical stimulation, although it was at amuch lower raterelative to normal heart rates. There was no significant differ-ence in the rates of this mechanically induced spontaneousheartbeat in Spca1�/� and Spca1�/� embryos (Fig. 6). Heartsof both genotypes had sufficiently developedmyofilaments (seebelow) and cell-cell contacts to produce coordinated cardiaccontractions, which progressed in waves from the primitive

FIGURE 5. Yolk sac, hematopoiesis, and vascular development inSpca1�/� and Spca1�/� embryos are ostensibly normal. A, H&E-stainedsections of yolk sac from Spca1�/� and Spca1�/� embryos revealed nodifferences in mesothelium (white arrows) or visceral endoderm (blackarrows), and nucleated red blood cells were abundant in blood islands ofboth genotypes (white arrowheads). B, nucleated red blood cells examinedby electron microscopy appeared normal in both wild-type (left) and nullmutant (right) embryos. N, nucleus. C, H&E-stained sections of wild-type(left) and null mutant (right) embryos revealed apparently normal dorsalaortae (black arrows) in both genotypes. The notochord (white arrow),mesenchyme (M), and oropharynx (P) are indicated.

FIGURE 6. Normal spontaneous cardiac contractions in Spca1�/� andSpca1�/� embryos. Spca1�/� and Spca1�/� embryos were removed from theyolk sac, and if no spontaneous contractions were apparent, the left ventricle wasmechanically stimulated. The frequency of the resultant rhythmic contractions(which does not correspond to the normal embryonic heart rate in utero) wasessentially the same for both genotypes at ED 9.5 (n � 13 �/� and 19 �/�embryos) and ED 10.5 (n � 2 �/� and 3 �/� embryos).

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ventricle through the outflow tract, propelling blood into thedorsal aortae. Red blood cells within the embryos were presentin sufficient numbers to be visible as a flow within both theheart tube anddorsal aortae, demonstrating that the circulatorysystem was intact.Presumptive myocardial and endocardial cells, trabeculae,

and division of the heart tube into appropriate segmentsoccurred in both genotypes (Fig. 7). Endocardial cushionsformed in the absence of SPCA1, since similar outflow tract

morphologywas seen in both Spca1�/� and Spca1�/� embryos(Fig. 7A). Heart development progressed with apparent nor-mality through ED 10.5; the looped heart, paired dorsal aortae,bulbous cordis, primitive ventricle, and common atrial cham-ber were all distinguishable. At higher resolution, the endocar-dium and trabeculae appeared similar in both wild-type andSpca1�/� hearts (Fig. 7B). Cardiac myocytes contained gly-cogen deposits by ED 9.5. Ultrastructurally, ED 10.5 myo-cytes of Spca1�/� and Spca1�/� hearts contained actin andmyosin filaments and exhibited clear evidence of banding,and both genotypes showed typical thin and thick filamentorganization in longitudinal sections and cross-sections(Fig. 7C).

FIGURE 7. Heart development appears normal in Spca1�/� embryos. A, H&E-stained sections of hearts from ED 9.5 Spca1�/� and Spca1�/� embryos.Measurements of endocardium, myocardial wall, and trabeculations revealed no apparent differences between the two genotypes, and development ofoutflow tract (OT) and atrioventricular (AV) cushions appeared normal. White arrows, myocardium; black arrows, cardiac cushions; bar, 100 �m. B, highermagnification (bar, 100 �m) of hearts at ED 9.5 shows that the myocardium (white arrows) and endocardium (black arrows) were similar in �/� and �/�embryos. The common ventricular chamber was filled with nucleated red blood cells in both genotypes (asterisks). C, electron microscopy of cardiacmyocytes from ED 10.5 embryos revealed apparently normal organization of the contractile apparatus; white arrows indicate Z bands; black arrowsindicate myofilaments; bar, 1 �m. As shown at higher magnification in the insets, thick (white arrowheads) and thin (black arrowheads) filamentsappeared well organized in both genotypes. Bar, 0.1 �m.

FIGURE 8. Morphological evidence of Golgi and ER stress in Spca1�/�

embryos. Electron micrographs of representative Spca1�/� (left) andSpca1�/� (right) cells illustrating differences in the Golgi and ER that occurredin the two genotypes. Highly ordered and closely stacked leaflets of the cen-tral lamellae of the Golgi apparatus (bracket and white arrow) were commonin wild-type embryos but tended to be dilated, less well organized, and moredifficult to identify in null mutant embryos. RER (black arrows) was oftendilated in null mutant cells and had reduced numbers of ribosomes. Densegranules (white arrowhead) were more common in mutant cells and wereincreased in numbers on the cis-side of the Golgi. Mitochondria (M) did notshow evidence of change. N, nuclei. Bar, 1 �m.

TABLE 1Morphometric analyses of the effects of SPCA1 ablation on Golgimembranes, Golgi-associated vesicles, and rough endoplasmicreticulumData were collected from electronmicrographs of ED 8.5–10.5 embryos. Values aremeans � S.E. n � 6–8 Spca1�/�, 6–11 Spca1�/� for each measurement.

8.5–10.5 ED �/� �/� pGolgi bodies/cell profile 0.08 � 0.02 0.40 � 0.15 0.012Golgi bodies (Vd)a 0.18 � 0.08 0.78 � 0.4 0.075Flat lamellae (number per Golgibody)

4.7 � 1.8 1.36 � 0.68 0.017

Dilated Golgi (percentage of totalGolgi)

44.4 � 20.5 89.1 � 4.2 0.010

Golgi-associated vesicles (Vd)b 0.51 � 0.16 1.57 � 0.64 0.007Golgi-associated dense vesicles/cellprofile

0.30 � 0.20 1.17 � 0.3 0.051

Dense vesicles on cis side (%) 3.2 � 2.8 42.7 � 11.5 0.016Rough ER (Vd) 2.52 � 0.30 2.62 � 0.49 0.83Dilated RER (percentage of total) 31.7 � 7.6 48.6 � 14.9 0.069Ribosomes/�m of RER 27.08 � 2.11 19.71 � 1.45 0.0001

aVd, volume density.b This Vdmeasurement largely excluded the small dense vesicles.

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Altered Golgi and Endoplasmic Reticulum in Null MutantEmbryos—Analysis of Spca1�/� embryos by electron micros-copy indicated that both Golgi membranes and rough endo-plasmic reticulum were altered (Fig. 8). The flattened, stackedlamellae typical of Golgi bodies had been lost or diminished inmost, if not all, tissues. The number of Golgi-associatedsmooth vesicles was increased in the mutant, and dense ves-icles associated with the Golgi appeared to have been sharplyincreased, particularly on the cis-side. Profiles of RER wereoften dilated, and the RER seemed to have diminished num-bers of ribosomes.

To quantify these changes, the numbers of these organellesper cell were counted, and/or their volume densities weredetermined (Table 1). Major alterations in Spca1�/� cellsincluded at least a 4-fold increase in Golgi bodies, dilation ofGolgi membranes, and significant changes in the number anddistribution of Golgi-associated vesicles. The amount of RERwas not altered, and the increase in dilated RER did not achievea high level of significance; however, the number of ribosomesper �m of RER was significantly reduced.

Given the changes in Golgi and RER membranes, one mightanticipate that structures dependent on processing and traf-ficking of proteins in the secretory pathway would be per-turbed. However, ultrastructural analysis of the neural tube,which was the only embryonic structure that was clearlyaltered, revealed normal-appearing coated pits, junctionalcomplexes, desmosomes, and basement membranes (Fig. 9).Ultrastructural analyses of other tissues, such as visceralendodermal cells, yielded similar results (data not shown).Morphometric analyses (Table 2) revealed no significant differ-ences in the numbers of coated pits, desmosomes, or junctionalcomplexes, and similar numbers of mitochondria were presentin both genotypes.Apoptosis and Intracellular Lipid Droplets Are Increased in

Null Mutant Embryos—The apparent growth retardationraised the possibility that Spca1�/� embryos on EDs 9.5 and10.5 might have a reduced rate of cell division and/or anincreased rate of apoptosis. While determining mitotic andapoptotic indices, a frequent observation in Spca1�/� cells wasthe apparent accumulation of lipid droplets within the cyto-plasm, whichwas confirmed by ultrastructural studies (Fig. 10).Therefore, a count of cytoplasmic lipid droplets was alsoconducted.Analysis of neural tube, mesenchyme, and the developing

cardiovascular system of wild-type and null embryos revealedno significant differences in mitotic index (Table 3). The apo-ptotic index, however, was significantly increased in both neu-

FIGURE 9. Ultrastructure of coated pits, junctional complexes, desmo-somes, and basement membranes appears normal in Spca1�/� embryos.Membrane specializations for cell adhesion appeared similar in cells fromSpca1�/� (left) and Spca1�/� (right) embryos. A, coated pits (white arrows)from neural tube cells. M, mitochondria. B, junctional complexes (whitearrows) from cells along the lumen of the neural tube. C, cytoplasmic denseplaques and intermediate filaments are clearly visible in desmosomes (whitearrows) from cells of both genotypes. D, basement membranes (arrows) ofneural tube cells. Bar, 0.1 �m for A and 1 �m for B–D.

FIGURE 10. Accumulation of excess lipid droplets in cells from Spca1�/�

embryos. A prominent change in Spca1�/� cells was the accumulation oflipid droplets, which were large enough to be counted by light microscopy in2-�m sections (see Table 3). Shown is an electron micrograph of a thin sectionshowing multiple large lipid droplets (L) in the cytoplasm of a cell from anSpca1�/� embryo.

TABLE 2Incidence of coated pits, desmosomes, junctional complexes, andmitochondriaData were collected from electron micrographs of ED 8.5–10.5 embryos. Values ofthe incidence per cell profile are means � S.E. n � 8 Spca1�/�, 11 Spca1�/�.

�/� �/� pCoated pits 0.11 � 0.03 0.07 � 0.03 0.82Desmosomes 0.037 � 0.026 0.051 � 0.031 0.46Junctional complexes 0.66 � 0.14 0.61 � 0.14 0.34Mitochondria 2.09 � 0.23 2.28 � 0.39 0.20

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ral tube and mesenchyme of Spca1�/� embryos. Head mesen-chyme, which included neural crest cells, was most adverselyaffected by loss of SPCA1. Apoptosis, accompanied by blebbingof the plasmalemma, fragmentation of the nuclear DNA, andproduction of dense apoptotic bodies, was a characteristic fea-ture of Spca1�/� mesenchyme and was particularly elevated inthe area of the trigeminal neural crest, which contributes to theproduction of a wide range of tissues. In contrast, neither mito-sis nor apoptosis was significantly altered in developing cardio-vascular tissues of Spca1�/� embryos. A prominent feature ofSpca1�/� tissues was a sharp increase in the numbers of cellswith more than three lipid droplets in the cytoplasm. Thisincrease was particularly apparent in the mesenchyme and inthe cardiovascular system.Squamous Cell Tumors in Adult SPCA1 Heterozygous

Mutants—When heterozygous mice were aged, they did notdevelop skin lesions characteristic of HHD; however, six of 26Spca1�/� mice developed squamous cell tumors of keratinizedepithelial cells (Table 4 and Fig. 11). These included three mice(20–22 months old) with carcinomas of the skin, a 24-month-

old mouse with a papilloma of the skin in the perianal regionand two 23-month-old mice with papillomas of the esophagus.Some of the carcinomas were quite large, with a 6.8-g tumor(15% of body weight) of the neck and interscapular region and a3.5-g tumor (7% of body weight) of the hind limb (Fig. 11B).None of the 23 similarly aged wild-type mice developed squa-mous cell tumors. Additional mice exhibited lung adenomas,lung adenocarcinomas, or liver adenomas, but the incidencewas essentially the same in wild-type and heterozygous mice.

DISCUSSION

Midgestational Death and Exencephaly in Spca1�/�

Embryos—The normal appearance of null embryos on ED 8.5showed that the loss of SPCA1 did not impair blastocyst devel-opment, implantation, formation of the three germ layers, orgastrulation. By ED 9.5, however, null embryos exhibitedgrowth retardation and exencephaly, and death occurredbetween ED 10 and ED 11. Themost common causes of embry-onic lethality during this period are failure of hematopoiesisand impaired development of the cardiovascular system (25).However, defects in yolk sac development or yolk sac-basedhematopoiesis (26, 27) were not responsible for the observedembryonic lethality. Similarly, none of the hallmarks ofimpaired cardiovascular development (28–31) occurred inSpca1�/� embryos. The only apparent structural defect wasincomplete closure of the neural tube, although there was somebending at the dorsolateral andmedial hinge points, suggestingthat signaling via factors secreted from the notocord that arerequired for neural plate bending (32) was not seriouslyimpaired. Neural tube defects have a diversity of causes (33),including apoptosis (34), which is the likely cause of thesedefects in Spca1�/� embryos.Embryolethality Correlates with Apoptosis and Secretory

Pathway Stress—The increased apoptosis suggests that embry-onic death was due to widespread failure of cell viability ratherthan the failure of any one cell type or organ system. Becausethe primary defect was the loss of SPCA1, it is clear that theapoptosis is due, directly or indirectly, to a deficiency in GolgiCa2� and/or Mn2� handling. In yeast, PMR1 null mutants arehighly sensitive to Mn2� toxicity, suggesting that one functionof the Mn2� transport activity of the pump is to expel Mn2�

from the cell via the exocytic pathway (35). Mn2� toxicity canoccur in humans and has been shown to cause apoptosis inmammalian cells (36); however, it is unclear that Mn2� detox-ification would be a critical function in a rapidly growingembryo. Because Mn2� is an important cofactor for glycosyl-transferases in the Golgi (37), impaired glycosylation of

FIGURE 11. Squamous cell papillomas and carcinomas in SPCA1 heterozy-gous mutant adults. A, papilloma of the esophagus isolated from a23-month-old Spca1�/� male; note the extensive keratinization (top). Anadditional focus of hyperplasia was located elsewhere in this esophagus.B, skin carcinoma from a 20-month-old Spca1�/� female located on the backof the right hind limb. Bar, 100 �m.

TABLE 3Morphometric analyses of the effects of SPCA1 ablation on mitotic index, apoptotic index, and the number of lipid inclusions in embryoniccellsData were collected fromH&E-stained sections from ED 8.5–10.5 embryos. Values are means � S.E. and represent the percentage of affected cells in that tissue. n � 8–12for each genotype.

Neural tube Mesenchyme Heart and vessels�/� �/� �/� �/� �/� �/�

Mitotic index 1.62 � 0.37 1.97 � 0.31 0.94 � 0.31 1.13 � 0.35 1.85 � 0.76 0.89 � 0.2Apoptotic index 0.42 � 0.18 2.91 � 1.38a 0.85 � 0.36 3.72 � 1.11a 0.40 � 0.3 1.19 � 0.63 lipid droplets/cell 0.08 � 0.06 3.27 � 1.36a 1.72 � 1.5 18.7 � 5.8a 1.31 � 0.6 19.2 � 7.1a

a p � 0.02.

TABLE 4Squamous cell carcinomas and papillomas in Spca1�/� miceSpca1�/� and Spca1�/� mice were aged for up to 2 years. Tumors of nonepithelialorigin did not differ significantly between the genotypes and were not included.

SkinEsophagus (papilloma)

Carcinoma Papilloma�/� (n � 26) 3 1 2�/� (n � 23) 0 0 0

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proteins could contribute to secretory pathway stress and apo-ptosis; however, Mn2� can enter mammalian secretory mem-branes via inositol 1,4,5-trisphosphate receptors (38), which arepresent in both the ER and Golgi (39). Thus, the more likelycause of the apoptosis phenotype is the defect in Golgi Ca2�

handling.A reduction in ERCa2� stores or inhibition of SERCA2 activ-

ity by thapsigargin is a well establishedmechanism for inducingER stress (40, 41), which leads to induction of the UPR andER-associated degradation (42, 43), with activation of both apo-ptotic and survival pathways. The Golgi also plays a role instress sensing and apoptotic signaling (44); however, well doc-umented conditions producing Golgi-specific stress have notbeen identified (44). The current study provides strong evi-dence that impaired Ca2� sequestration by SPCA1 can causeGolgi-specific stress, which seems analogous to ER-specificstress resulting from SERCA2 deficiency.Major ultrastructural manifestations of Golgi stress included

dilation of the Golgi and a reduction in the number of stackedleaflets. An increase in Golgi membranes and Golgi-associatedvesicles was also observed and, as discussed below, may be aresponse to Golgi stress that allows cell survival. Dilation ofGolgi membranes and expansion of the Golgi have beenobserved both in human Tangier disease and in mice lackingATP-binding cassette transporter 1 (45, 46), which is expressedin both Golgi and plasma membranes (47). The cytoplasmiclipid droplets in Spca1�/� cells may also be due to Golgi stress,since the Golgi plays a critical role in lipid metabolism andtrafficking (48).There was some dilation of the RER and a significant

decrease in the number of membrane-bound ribosomes, con-sistent with increased accumulation of protein and reducedprotein synthesis that occurs during ER stress (41, 49). Thesharp increase in the number of dense vesicles on the cis-side ofthe Golgi suggests that trafficking of vesicles between the ERandGolgi was affected. The effects of SPCA1 null mutations onthe ER are consistent with observations that a reduction inSPCA1 expression in cultured cells causes hypersensitivity toER stress and impairs ER-associated protein degradation (12).Expansion of the Golgi as a Possible Survival Response to

Golgi Stress—Despite increased apoptosis and Golgi stress, theviability of Spca1�/� embryos during earlier stages of embryo-genesis and the normal development of coated pits, desmo-somes, junctional complexes, and basementmembranes, whichwould require a functional secretory pathway, shows that theloss of SPCA1 does not cause a massive failure of Golgi func-tion. That a substantial degree of Golgi function is retainedsuggests that at least partial compensation occurs for any func-tional deficits caused by the loss of SPCA1. It is unlikely thatSPCA2 is involved, since it is expressed in only a limited set oftissues (5, 6), and reverse transcription-PCR analysis showedthat its mRNA is not expressed in ED 8.5 embryos (data notshown). Other means by which delivery of Ca2� to the Golgimight occur is by the activity of a thapsigargin-sensitive Ca2�

pump (presumably SERCA2) that mediates Ca2� uptake intoan agonist-sensitive compartment of the Golgi (8) and by traf-ficking of Ca2�-containing vesicles from the ER to the Golgi.

The �4-fold expansion of the Golgi in Spca1�/� embryoswould appear to be an important compensatory mechanism,since it should improve the overall capacity for processing andtrafficking of proteins in the secretory pathway. We are awareof only one other documented example of Golgi expansion in adisease state (45, 46); however, expansion of the ER occurs dur-ing differentiation and in response to ER stress resulting from avariety of conditions (reviewed in Ref. 50). The transcriptionfactor XBP1, which is activated in response to ER stress and isan effector of the survival pathways of the UPR (42), induces ERbiogenesis (51, 52). Overexpression of XBP1 leads to inductionof genes encoding proteins of multiple compartments of thesecretory pathway, including theGolgi (53), and to expansion ofthe Golgi itself (54). Thus, it is possible that expansion of theGolgi in Spca1�/� embryos is elicited by survival pathways acti-vated in response to Golgi stress, which are directly analogousto (and may interact or overlap with) survival pathways acti-vated in response to ER stress.Squamous Cell Tumors in SPCA1 Heterozygous Mice—

Spca1�/� mice exhibited no manifestations of HHD but diddevelop squamous cell carcinomas and papillomas. The pheno-type is similar to that of SERCA2 heterozygous mice, whichdeveloped squamous cell tumors but notDarier disease (17, 18).Thus, the Spca1�/� mouse is the second genetic model of can-cer involving a deficiency in Ca2� transport. Because SPCA1,like SERCA2, functions in the secretory pathway but playsmuch less of a role in Ca2� signaling than SERCA2, it is likelythat impairments of the secretory pathway itself is part of themechanism of tumorigenesis in both models.The incidence of squamous cell tumors was �25% in

Spca1�/� mice, compared with �90% among SERCA2 het-erozygous mutants (17, 18). The normal incidence is very low;e.g. among 4900 control mice in the National Toxicology Pro-gram studies (55), only one esophageal and 11 skin squamouscell tumors were observed, six of which were carcinomas.Tumors in SERCA2 heterozygous mice did not exhibit loss ofthe wild-type allele (18), thereby ruling out the possibility thatSERCA2 serves as a classical tumor suppressor inmice. The lateage of onset makes it unlikely that loss of heterozygosity is partof the genetic mechanism of tumorigenesis in Spca1�/� mice.In humans, there is at least one case of type 2 segmentalHHD inwhich loss of heterozygosity occurred at the ATP2C1 (SPCA1)locus during embryonic development, leading to mosaicismand severe HHD in SPCA1 null cells (56). The lack of skintumors in this patient shows that SPCA1 does not serve as aclassical tumor suppressor in humans.Similarities between Phenotypic Effects of SPCA1 and

SERCA2 Deficiency Suggest a Common Mechanism—Despitemajor species differences in disease phenotypes, there are clearparallels between the effects of SPCA1 and SERCA2 heterozy-gosity in humans and mice. For each Ca2� pump and for bothspecies, the affected cell type is keratinocytes, and the site of theprimary deficit in Ca2� handling is a compartment of the secre-tory pathway. In humans, loss of one copy of either gene causesacantholytic skin disease (13, 14, 16), whereas squamous celltumors, with no evidence of skin disease, are the major pheno-type inmice (17, 18) (this study). These similarities suggest thatthe mechanisms of each disease are similar. However, why

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would reduced activity of either SPCA1 or SERCA2 cause squa-mous cell tumors in mice and acantholytic skin disease inhumans? The answer is undoubtedly complex but may involvedifferential responses of mouse and human keratinocytes tosecretory pathway stress, with prosurvival pathways that favortumorigenesis predominating in mice and proapoptotic path-ways that favor acantholysis predominating in humans. In con-sidering this possibility, which is admittedly speculative, someuncertainties should be noted. Although a great deal is knownabout ER stress responses, almost nothing is known aboutGolgistress responses other than the observations that severe Golgistress can affect ER function, induceGolgi expansion (an appar-ent prosurvival response), and induce apoptosis.Conditions causing deficiencies in protein processing and

trafficking in the secretory pathway, including nutrient depri-vation, hypoxia, exposure to certain anti-tumor agents, or areduction in luminal Ca2� concentrations, can cause ER stress(57). This leads to induction of the UPR, a set of powerful stressresponse pathways that activate both prosurvival and proapo-ptotic mechanisms. Severe ER stress, to which the cell cannotadapt, leads to apoptosis; however, activation of the UPR bychronic low level ER stress leads to adaptation, since prosur-vival stimuli remain active and proapoptotic stimuli diminish(58). The major effectors of the UPR are the transcription fac-tors XBP1 andATF6, and the kinase PERK, which affects trans-lation (41, 59). Thus, the UPR causes major alterations inmRNA and protein expression patterns that either enable thecell to survive or, alternatively, lead to death.Although the details are not well understood, the role of the

UPR in tumor survival and progression is well established (60),with both XBP1 and PERK playing prominent roles (61, 62).Activation of the UPR by exposure to conditions causing ERstress can lead to protection against ER stress caused by thesame or different conditions (58). That mild SERCA2 defi-ciency has the potential to cause ER stress and induce adaptivecomponents of the UPR is supported by the observation thattreatment of cultured cells with very low concentrations of theSERCA inhibitor thapsigargin leads to sustained activation ofprosurvival pathways of the UPR with only transient activationof proapoptotic pathways (58). Thus, it is possible that sus-tained activation of prosurvival pathways of cellular responsesto ER or Golgi stress might play a mechanistic role in tumordevelopment in SERCA2 and SPCA1 heterozygous mice.In humans, it has been suggested that HHD and Darier dis-

ease may result from defective processing and trafficking ofdesmosomal components (63). In the case of HHD, this mech-anism seems unlikely, since desmosomes and other cell surfacestructures appeared normal in null mutant mice. Also, in apatient with type 2 segmental HHD, in which both copies of theATP2C1 (SPCA1) gene were defective (56), some of the genet-ically affected skin appeared normal. One might expect that ifheterozygosity caused a sustained defect in trafficking andprocessing of desmosomal proteins, then homozygous nullmutations would cause a massive failure. Type 2 segmentalHHDwasmore severe and affected deeper layers of the skin butwas still recognized asHHD, suggesting that the disease processwas fundamentally the same as in heterozygous HHD. Duringdifferentiation, keratinocytes undergo a highly controlled proc-

ess of “incomplete” apoptosis (64–67) in which they lose theirnuclei and die but retain the intercellular connections andintracellular keratin intermediate filaments that provide struc-tural integrity to the skin. Perhaps the loss of one copy of eithergene causes ER or Golgi stress, which in turn elicits responsesthat activate proapoptotic pathways that interfere with the reg-ulation of incomplete apoptosis needed for terminal differenti-ation. Ultrastructural changes in cells from Darier diseaselesions (68) are consistent with aberrant apoptosis as part of thedisease mechanism. Desmosomal proteins are targeted duringapoptosis (69, 70), so if the incomplete apoptosis that occursduring normal keratinocyte differentiation in humans wereaccentuated by additional apoptotic stimuli, breakdown of des-mosomal contacts between cells and their attachments to inter-mediate filaments, one of the hallmarks of HHD and Darierdisease, could occur.Conclusions—Our findings establish SPCA1deficiency as the

first documented example of a Golgi-specific stress conditionand identify Spca1�/� mice as the second genetic model inwhich a deficiency in Ca2� handling causes cancer. When con-sidered in the light of previous studies, a number of interestingparallels between the phenotypic effects of SPCA1 and SERCA2haploinsufficiency are apparent. A reduction in the activity ofeither Ca2� pump, each of which serves in the secretory path-way, caused squamous cell tumors in mice and acantholyticskin disease in humans, and, in each case, keratinocytes werethe affected cell type. This suggests a common disease mecha-nism in which 1) chronic secretory pathway stress, originatingin either the ER or Golgi, leads to 2) activation of cellular stressresponses, such as the UPR or some comparable Golgi-specificstress response (about which almost nothing is known), and 3)differential activities of prosurvival or proapoptotic arms ofthe stress response in mice or humans, respectively, leads tothe observed disease manifestations in each species. If thisview is correct, then the prosurvival and proapoptotic stressresponses themselves serve as major components of the dis-ease mechanism.

Acknowledgments—We thank Dr. Tim Reinhardt (National AnimalDisease Center, Ames, IA) for providing the SPCA1 antibody andMaureen Bender for expert animal husbandry.

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SPCA1 Gene Targeting

SEPTEMBER 7, 2007 • VOLUME 282 • NUMBER 36 JOURNAL OF BIOLOGICAL CHEMISTRY 26527

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