lossoftheatp2c1secretorypathwayca2 -atpase(spca1 ... ?· spca1, causes hailey-hailey disease...
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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. Shull1
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 (13), 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 Ca2andMn2 (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 Ca2pump 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: email@example.com.
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. 2651726527, September 7, 2007 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
SEPTEMBER 7, 2007 VOLUME 282 NUMBER 36 JOURNAL OF BIOLOGICAL CHEMISTRY 26517
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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.
Generation of Mutant MiceA 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 182228 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 AnalysisGenotyping 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 475501and 725750 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 mRNAsTotal 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 182190 in exon 8 and the reverse complement ofcodons 428435 in exon 15. For embryos, PCR primers corre-sponded to codons 182190 and the reverse complements ofeither codons 366373 in exon 13 or codons 660667 in exon
21. PCR products were analyzed by agarose gel electrophoresisand DNA sequencing.Immunoblot Analysis of SPCA1ProteinNewborn 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 at80 C until further use.Epithelial samples were manually homogenized as describedbefore (17). Adult mouse hearts were manually pulve