Human Keratinocyte ATP2C1 Localizes to the Golgi and Controls Golgi Ca2+ Stores

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Title Human keratinocyte ATP2C1 localizes to the Golgi and controls Golgi Ca2+ stores

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Journal Journal of Investigative Dermatology, 121(4)

ISSN 0022-202X

Authors Behne, M J Tu, Chia-Ling L Aronchik, I et al.

Publication Date 2003-10-01

Peer reviewed

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Human Keratinocyte ATP2C1 Localizes to the Golgi and Controls Golgi Ca2 þ Stores

Martin J. Behne,Ãw Chia-Ling Tu,z Ida Aronchik,w Ervin Epstein,Ãy Graham Bench,8 Daniel D. Bikle,z Tullio Pozzan,z and Theodora M. MauroÃw ÃDepartment of Dermatology, University of California, San Francisco, California, USA; wDermatology Service and zDepartment of Medicine, Endocrine Unit,VA Medical Center, San Francisco, California, USA; yDepartment of Dermatology, San Francisco General Hospital, San Francisco, California, USA; 8Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory, Livermore, California, USA; zDepartment of Biomedical Sciences and Consiglio Nazionale delle Ricerche (CNR) Center for the Study of Biomembranes, University of Padova, 35121 Padova, Italy

Hailey^Hailey disease (MIM16960) is a blistering skin parable to other epithelial cells, Hailey^Hailey disease disease caused by mutations in the Ca2 þ ATPase keratinocyte Golgi Ca2 þ re¢ll is slower, and the maxi- ATP2C1. We found that the abnormal Ca2 þ signaling mum Ca2 þ concentration reached is signi¢cantly lower. seen in Hailey^Hailey disease keratinocytes correlates with These ¢ndings were replicated in vivo, because clinically decreased protein levels of ATP2C1. Human ATP2C1 normal Hailey^Hailey disease epidermis contained low- protein approximated 115 kDa in size. The ATP2C1 er Ca2 þ stores and displayed an abnormal Ca2 þ gradi- is localized to the Golgi apparatus in human keratino- ent. In this report we localize the ATP2C1, demonstrate cytes, similar to its localization in yeast and Caenorhab- its physiologic relevance in mammalian cells, and ditis elegans. To test whether the ATP2C1 controls Golgi measure intraorganelle Golgi Ca2 þ in keratinocytes. Ca2 þ stores, we measured intraorganelle Ca2 þ concen- Key words: Hailey^Hailey disease/aequorin/adhesion/epider- trations using speci¢cally targeted aequorins. Whereas mal permeability barrier. J Invest Dermatol 121:688 ^694, 2003 normal keratinocytes display Golgi Ca2 þ levels com-

ailey-Hailey disease (HHD) is an autosomal domi- (Hu et al, 2000), suggesting that ATP2C1 controls a Ca2 þ store nant blistering skin disease, characterized histo- that is important in the keratinocyte response to raised extracellu- logically by defects in cell-to-cell adhesion in lar Ca2 þ. the suprabasal layers of the epidermis (acantholysis). Although the central role of the PMR1 and Golgi calcium HHD is a result of mutations in ATP2C1 (Hu et al, stores is well established in yeast (Sorin et al, 1997; Durr et al, H2000; Sudbrak et al,2000),aCa2 þ ATPase localized to the Golgi 1998), the discovery that ATP2C1 mutations caused defects in in Caenorhabditis elegans (Van Baelen et al, 2001) and whose homo- both intracellular Ca2 þ sequestration and intracellular Ca2 þ sig- log, PMR1, also is localized to the Golgi in yeast (Antebi and naling, and resulted in human disease, was the ¢rst indication of Fink, 1992). Histologic and immuno£uorescence studies of the importance of this Ca2 þ ATPase in mammals (Hu et al,2000; HHD demonstrate internalization of desmosomal components, Sudbrak et al, 2000). Mutations found in HHD patients most of- whereas adherens junctions and gap junctions are preserved (Har- ten predict prematurely truncated products through frameshift ada et al, 1994; Hashimoto et al, 1995; Metze et al, 1996). In normal mutations or single-base-pair substitutions, consistent with a keratinocytes in vitro,Ca2 þ stimulates the assembly of functional haploinsu⁄ciency pathogenesis (Hu et al, 2000). Endoplasmic re- desmosomes, both by causing the formation of actin ¢lopodia ticulum (ER) calcium stores also are present in keratinocytes, and and by complexing desmosomal components including E-cad- pharmacologic blockade of the Ca2 þ ATPase ATP2A2 with herin, a-, b-, and g-catenin (Vasioukhin et al,2000).Raisedex- thapsigargin (TG) prevents Ca2 þ -induced redistribution of E- tracellular Ca2 þ acts through a well-de¢ned cascade of signaling cadherin to the cell periphery (Li et al, 1995). Mutations in the events, including binding to a plasma membrane Ca2 þ receptor, ATP2A2 cause Darier’s disease (Sakuntabhai et al, 1999), a similar generation of inositol 1,4,5-trisphosphate (IP3), release of intracel- blistering skin disease. In contrast, the role of ATP2C1-controlled lular Ca2 þ, and subsequent in£ux of Ca2 þ through plasma Golgi Ca2 þ stores in keratinocyte Ca2 þ -sensitive processes such membrane channels. HHD keratinocytes do not release intra- as growth, di¡erentiation, and adhesion has not yet been de¢ned. cellular Ca2 þ when stimulated with raised extracellular Ca2 þ The yeast homolog to the ATP2C1, PMR1, localizes to the medial Golgi (Sorin et al, 1997) and can raise Golgi Ca2 þ concentrations to levels as high as 1 mM (Taylor et al, 1997). In yeast, Manuscript received November 5, 2002; revised February 17, 2003; intraorganelle Ca2 þ controls Golgi transport (Carnell and accepted for publication May 12, 2003 Moore, 1994), N-linked glycosylation, accurate sorting of car- Reprint requests to: Martin J. Behne, Dermatology Service (190), Veter- Ã ans A¡airs Medical Center, 4150 Clement Street, San Francisco, CA 94121. boxypeptidase Y, and the appropriate degradation of Cyp , Email: [email protected] a misfolded soluble ER protein (Durr et al, 1998). PMR1 mutants are intolerant to raised extracellular Ca2 þ and demonstrate Abbreviations: ER, endoplasmic reticulum; HHD, Hailey^Hailey dis- 2 þ ease; IP3, inositol 1,4,5-trisphosphate KRB, Krebs^Ringer bicarbonate; higher cytosolic Ca concentrations (Halachmi and Eilam, TG, thapsigargin; PIXE, proton-induced X-ray emission (analysis). 1996), similar to that seen in HHD keratinocytes (Hu et al,2000).

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688 VOL. 121, NO. 4 OCTOBER 2003 ATP2C1 CONTROLS GOLGI CA2 þ 689

Experiments demonstrating TG-sensitive and -insensitive cellular Ca2 þ. Calibration was performed at the end of each experiment by lysing þ stores suggested that a Ca2 ATPase distinct from the ATP2A2 the cells with a solution containing 100 mM digitonin and 10 mM CaCl2 in 2 þ H O. The photomultiplier output was ampli¢ed by a built-in ampli¢er^ was linked to IP3 -sensitive Ca pools (Pizzo et al, 1997). These 2 ¢ndings were con¢rmed and expanded by the discovery that the discriminator. This signal was captured by a Thorn-EMI photon counting board and stored in an IBM-compatible computer for further analysis. The homologous PMR1 ortholog expressed in C. elegans controls an 2 þ 2 þ resulting luminescence data were calibrated into Ca values, using a IP3 -sensitive Ca store located in the Golgi (Van Baelen et al, computer algorithm (Brini et al, 1995). 2001). Human ATP2C1 is homologous but not identical to yeast PMR1 (Rudolph et al, 1989) and encodes two alternatively spliced transcripts, ATP2C1a and ATP2C1b. Cellular fractionation (sucrose gradient) Normal keratinocytes 2 þ were cultured as above. The cells were homogenized using a Polytron Unlike measurements of cytosolic Ca , Golgi intraorganelle homogenizer, the nuclei removed by 500 g centrifugation, and crude 2 þ 2 þ Ca cannot be measured accurately with Ca -sensitive dyes. membranes were collected by high-speed centrifugation at 10,000 g. The Therefore, we used a targeted aequorin approach similar to that resulting supernatant was layered on a discontinuous, multilayer sucrose used to study the Ca2 þ concentrations in mitochondria (Rizzuto gradient (15%^50%), centrifuged in a Beckman SW28 rotor at 100,000 et al, 1992), nucleus (Brini et al, 1993), and ER (Montero et al,1995). g for 16 h, and the membranous materials in the various sucrose layers We transfected normal and HHD keratinocytes with a chimeric collected for western blot analysis. Organelle and ATP2C1 localization was cDNA that encodes an HA1-tagged aequorin (Brini et al,1995) accomplished using antibodies to BiP (mouse monoclonal antibody and the transmembrane portion of sialyltransferase, which targets 610978, BD Transduction Laboratories, San Diego, CA) as the ER marker, the aequorin chimera to the Golgi (Pinton et al,1998).Thistool, GM130 as the cis-Golgi marker (mouse monoclonal antibody 610822, BD - Transduction Laboratories), p230 (mouse monoclonal antibody 611280, BD and its related aequorin mutant (Asp119 Ala), which converts Transduction Laboratories), and TGN38 (mouse monoclonal antibody the aequorin to one with a lower Ca2 þ a⁄nity and thus a more 2 þ 610898, BD Transduction Laboratories) as the trans-Golgi markers and long-lasting signal at micromolar Ca concentrations, has been ATP2C1 (PMR1, rabbit polyclonal IgG, sc-5548, Santa Cruz Bio- used previously to measure Golgi Ca2 þ concentrations in normal technology, Santa Cruz, CA) as well as ATP2A2 (SERCA2, sc-8094, and Bcl-2-transfected HeLa cells (Pinton et al, 1998; Pinton et al, Santa Cruz Biotechnology). 2000). Using this approach, we found that normal human kerati- 2 þ nocytes also sequester substantial Ca pools into the Golgi ap- Immunohistochemistry Normal keratinocytes, cultured as described paratus and that these stores are depleted in keratinocytes and above, were ¢xed with 4% formaldehyde in phosphate-bu¡ered saline for epidermis after mutation of the ATP2C1. Further, we have loca- 10 min. ATP2C1 was labeled using a rabbit polyclonal antibody to human lized the ATP2C1 protein to the Golgi apparatus in keratinocytes, ATP2C1 (sc-5548, Santa Cruz Biotechnology). The same cells also were using cellular fractionation and functional Ca2 þ concentration labeled with the trans-Golgi membrane-associated protein p230 (mouse studies. Finally, we demonstrated that this defect in intracellular monoclonal antibody 611280, BD Transduction Laboratories) or the cis- Ca2 þ regulation is manifested in the epidermis by a reduction in Golgi matrix protein GM130 (mouse monoclonal antibody 610822, BD 2 þ Transduction Laboratories). Secondary antibodies were FITC^goat anti- the Ca gradient critical for normal epidermal di¡erentiation rabbit (Jackson ImmunoResearch, No. 111-096-046) and Texas red^ and barrier function. donkey anti-mouse (Jackson ImmunoResearch, No. 715-075-151). All primary antibody incubations were performed at 41C overnight and diluted 1:100, and secondary antibodies were used at 1:200 for 2 h at room MATERIALS AND METHODS temperature. Digital images were taken on a Zeiss 510 confocal microscope, using a 63 lens. Negative controls omitting the primary antibodies HHD patient selection All patients studied were diagnosed with HHD demonstrated equal to background nonspeci¢c staining. before entry into the study, demonstrating typical clinical ¢ndings (blistering and erosions in characteristic locations) and histologic Western immunoblotting Western immunoblotting was performed pathology (acantholysis of suprabasal cells without apoptosis). All patients using 8.5% SDS^PAGE, as described previously (Laemmli, 1970). Following signed consent forms in a study approved by the University of California, transfer of protein to PVDF membranes and incubation overnight with the San Francisco, Committee on Human Research. Normal human primaryATP2C1 antibody at a 1:1000 dilution at 4 1C (rabbit polyclonal IgG, keratinocytes, matched to age, location of skin biopsy (trunk), and pass sc-5548, Santa Cruz Biotechnology) and secondary antibody at room number were used as controls. Cells from four patients were studied in temperature for 2 h (peroxidase-conjugated anti-rabbit; Amersham this report. One patient had been analyzed in a previous report (Hu et al, Pharmacia Biotech Inc., Piscataway, NJ), ¢nal detection was performed by 2000) and su¡ered from a non-sense mutation in exon 16, which predicts chemiluminescence (Amersham). The cells were thawed and homogenized an abnormal, truncated protein. Cells from this patient were used for some þ by soni¢cation, and the protein contents were determined using the BCA of the aequorin Golgi Ca2 measurements. kit (No. 23227, Pierce, Rockford, IL), and individual lanes were loaded with volumes containing 30 mg of sample for subsequent SDS^PAGE analysis. Cell culture and transfection Second- to ¢fth-passage cultured human Equal loading per sample was controlled via individual protein content keratinocytes from adult normal skin (surgical skin margins) (cultured analysis and colloidal Coomassie brilliant blue staining (LC6025, Invitrogen adult human keratinocytes) or clinically normal HHD skin (punch Life Technologies, Carlsbad, CA) of the SDS gels following transfer to PVDF biopsies) were grown on 60-mm dishes, chamberslides, or glass coverslips 2 þ membranes. Additionally, densitometry was performed on the ¢nal chemi- in 0.06 mM Ca Epilife medium (Cascade Biologics, Eugene, OR) until luminescence images, using the Bio-Rad GS-710 scanner and Quantity One approximately 50% con£uence for immunohistochemistry, 100% con- analysis software. Optical density values were ¢rst adjusted to average £uence for aequorin measurements, sucrose gradient studies, and Western background density of the ¢lm, and normalization was achieved by immunoblotting. Twenty-four to forty-eight hours before aequorin mea- reprobing the same PVDF membrane with an anti-b-actin antibody (clone surements, 60% to 80% con£uent keratinocytes were transfected with AC-74, Sigma, St. Louis, MO). Expression values were normalized to actin 1 mg of GoAEQmut DNA per 2 mL TransIT keratinocyte transfection re- within same samples, and a percentage value for the di¡erence between agent (PanVera Corp., Madison,WI) per 1 mL of medium. After overnight conditions was calculated. Molecular sizes were calculated by a regression incubation, the cells were washed with phosphate-bu¡ered saline and analysis based on the prestained color standards routinely used for PAGE. returned to the Epilife medium.

Aequorin measurements Transfected normal and HHD keratinocytes Proton-induced X-ray emission PIXE analysis was performed using a were depleted of their intracellular Ca2 þ stores by incubation in Krebs^ modi¢cation of Bunse et al (1991). Clinically normal trunk skin biopsies Ringer bicarbonate (KRB) medium containing 125 mM NaCl, 5 mM from normal and HHD patients were snap-frozen in liquid propane/ KCl, 1 mM Na3PO4, 1 mM MgSO4, 5.5 mM glucose, 1 mM ionomycin, liquid nitrogen. Thirty-micrometer sections were transferred to a metal- and 500 mM EGTA. Cells were incubated for 1 h at 41C, conditions free nylon foil and freeze-dried for 12 h at ^801C. Data were obtained previously shown not to change the morphology of the Golgi (Pinton et using 3-MeV proton beams, a 0.8-mA current, and a 5-mm spot diameter, al, 1998). The coverslips were placed in close proximity to a low-noise with a scan size of 300 30 mm and binned into 10-mm segments for photomultiplier, in a 41C chamber, perfused ¢rst with KRB medium analysis. X-rays were detected with a Si(Li) detector, located at an angle containing 2% bovine serum albumin and 1 mM EGTA to wash the cells, of 1351 with respect to the incident beam, that subtended a solid angle of then with KRB plus 500 mM EGTA, and ¢nally with KRB plus 0.5 mM B100 msr. After PIXE analysis, the samples were counterstained with 690 BEHNE ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY hematoxylin and eosin, and epidermal thickness was measured using a et al, 1995; Pinton et al, 1998) and thus measures Golgi Ca2 þ lens micrometer. Data were reduced o¡-line so that X-ray spectra from speci¢cally. In organelles with an elevated Ca2 þ concentration, subregions could be extracted from each irradiated region. X-ray spectra such as ER or Golgi, Ca2 þ measurements using coelenterazine were analyzed with the PIXE spectrum ¢tting code (Antolak and Bench, 2 þ þ tend to underestimate the true Ca concentration, owing to 1994). Thin-¢lm calibration standards containing Ca2 were used to measure the e⁄ciency of the X-ray. Each sample was measured in three rapid consumption (Pinton et al, 1998). Therefore, we substituted separate areas. Data are presented as the mean7SD. the coelenterazine analog, coelenterazine N, which reduces the rate of aequorin consumption and restored Ca2 þ stores only at the time of the experiments (Barrero et al, 1997). Normal RESULTS keratinocytes were transfected with GoAEQmut, depleted of their Ca2 þ stores using 1 mM ionomycin, and then incubated The ATP2C1 protein localizes to the Golgi in keratino- with 5 mM coelenterazine N. Experiments using normal cytes The C. elegans ATP2C1 and its yeast homolog PMR1 keratinocytes demonstrated that store re¢lling was optimal using þ both are found in the Golgi apparatus (Antebi and Fink, 1992; extracellular Ca2 concentrations from 0.5 to 2 mM (data not þ Van Baelen et al, 2001). Using an antibody raised to human shown). Because raising extracellular Ca2 also can trigger þ ATP2C1 (PMR1, Santa Cruz Biotechnology Inc.), we localized intracellular Ca2 release (Varani et al, 1995; Bikle et al,1996; the human ATP2C1 to the keratinocyte Golgi apparatus (Fig 1). Hu et al, 2000) in human keratinocytes, we used the lowest þ Keratinocyte membranes were fractionated with a discontinuous concentration that re¢lled intracellular Ca2 stores, 0.5 mM þ þ sucrose gradient (15, 25, 35, 45, and 50% sucrose) and analyzed for Ca2 , for our baseline measurements. When Ca2 stores were þ colocalization of markers of subcellular organelles and ATP2C1. restored by perfusion with 0.5 mM extracellular Ca2 (Fig 3a), þ Markers of the ER (BiP) were found primarily in the lighter Golgi Ca2 in normal keratinocytes rapidly increased to fractions (15 and 25% sucrose) as were markers of the cis-Golgi 241725.1 mM (range 215^292 mM, n ¼ 8), a concentration (GM130). The trans-Golgi markers (p230, TGN38) ran in the slightly lower than measured in HeLa cells (Pinton et al,1998) heaviest fractions (35%^50% sucrose). ATP2C1 was found in and 1000-fold higher than found in cytoplasm. þ the trans-Golgi fractions. As expected, the ER calcium pump, Next, to test whether the ATP2C1 controls Golgi Ca2 ,we þ ATP2A2 (SERCA2), was found in the ER fractions. ATP2C1 compared Golgi Ca2 concentrations in normal versus HHD þ also was localized to the trans-Golgi using immunostaining, as keratinocytes. Golgi Ca2 stores re¢lled more slowly in HHD þ the localization of the ATP2C1 and the trans-Golgi marker p- keratinocytes, and the maximum Ca2 concentration reached 230 completely overlapped (Fig 1B). Although there was some was markedly lower, 150.6723.2 mM(n¼ 9; po001) (Fig 3b). overlap of the ATP2C1 and the cis-Golgi marker GM-130, the HHD keratinocytes for these experiments were derived from þ perinuclear localization of the ATP2C1 seemed to extend three patients, and Golgi Ca2 re¢lling was variable (range 95^ beyond the punctate staining of the GM-130 (Fig 1B). Thus, the 173 mM), probably re£ecting di¡ering degrees of ATP2C1 ATP2C1 is localized to the trans-Golgi both by sucrose gradient impairment among di¡erent subjects, as has been seen in and by immunohistochemistry techniques. attempts to correlate molecular and clinical ¢ndings in HHD patients (Dobson-Stone et al, 2002). These ¢ndings, coupled with The ATP2C1 protein is decreased in HHD keratinocytes our previous ¢ndings of increased cytosolic Ca2 þ in HHD Previous studies described decreased function of the ATP2C1 in keratinocytes (Hu et al, 2000), are best explained by a defective HHD keratinocytes (Hu et al, 2000). To test whether impaired intracellular Golgi Ca2 þ ATPase, leading to both decreased function resulted from a decrease in ATP2C1 protein, we Golgi Ca2 þ stores and increased cytosolic Ca2 þ. compared levels of ATP2C1 protein in normal versus HHD keratinocytes. ATP2C1a is predicted to encode 919 amino acids The epidermal Ca2 þ gradient is dissipated in HHD Finally, (Hu et al, 2000), with a predicted weight of 117 kDa, whereas to determine the location and proportion of ATP2C1-controlled ATP2C1b is predicted to encode 888 amino acids (Hu et al, Ca2 þ concentrations to total Ca2 þ concentrations, we compared 2000), with a predicted weight of 113 kDa. ATP2C1 protein total Ca2 þ concentrations in normal versus HHD keratinocytes measured approximately 115 kDa (Fig 2A,B). Further, ATP2C1 and epidermis. We had found previously that total Ca2 þ stores protein was reduced markedly in HHD keratinocytes (Fig were decreased by B40% in HHD cultured keratinocytes, and 2A,B). Because acantholysis in HHD is found in suprabasal that total epidermal calcium, measured by PIXE, was decreased layers (i.e., more di¡erentiated keratinocytes), initial comparisons in HHD epidermis (Hu et al, 2000). To localize di¡erences were performed on a total of six cell preparations from three between normal and HHD keratinocytes within the epidermis, patients, in cells grown in 1.2 mM Ca2 þ (a typical example is and to determine whether decreased skin Ca2 þ concentrations shown in Fig 2A). Expression was decreased in all samples and were limited to the epidermis, we next used PIXE to compare averaged 53.5% of normal (719.6 SD, range 25.5%^78%, n ¼ 6, Ca2 þ in normal versus HHD skin. We found that total Ca2 þ p ¼ 0.001, as assessed in a two-tailed, paired Student’s t test). concentrations were decreased substantially in normal HHD Moreover, ATP2C1 expression was decreased both in un- epidermis, whereas the dermis (deeper than B125 mm) showed di¡erentiated and in di¡erentiated keratinocytes (Fig 2B,D). no di¡erence in Ca2 þ concentrations between normal and These experiments suggest that the HHD phenotype results HHD (Fig 4A). Further, we found that total epidermal Ca2 þ from decreased levels of the ATP2C1 protein. did not di¡er in basal keratinocytes. This was surprising, because we found that undi¡erentiated keratinocytes in vitro display The Golgi contains a signi¢cant Ca2 þ store, controlled by marked di¡erences in intracellular Ca2 þ stores (Hu et al,2000; the ATP2C1 Total Ca2 þ stores in HHD are decreased, even and see Fig 3). Because ATP2C1 controls an intracellular Ca2 þ though ER Ca2 þ stores controlled by the ATP2A2 are normal store, these data suggest either that intracellular Ca2 þ stores (Hu et al, 2000). Additionally, although Golgi Ca2 þ constitutes a represent a relatively small percentage of total epidermal Ca2 þ signi¢cant Ca2 þ store in yeast and HeLa cells, the relative con- in the basal layer or that other cellular mechanisms success- tribution of the ATP2C1-controlled Golgi Ca2 þ to total fully compensate for the loss of ATP2C1 in basal layer but Ca2 þ stores in the keratinocyte is unknown. We used several not spinous or granular layer keratinocytes. The epidermal approaches to determine the magnitude and regulation of Ca2 þ gradient might also dissipate if the epidermal permea- ATP2C1-controlled Ca2 þ stores. First, we measured Golgi intra- bility barrier is damaged (Mauro et al, 1998). Nevertheless, organelle Ca2 þ concentrations in normal keratinocytes, using the HHD biopsy was taken from clinically normal skin. More an aequorin construct targeted speci¢cally to the Golgi appara- importantly, gradients in other ions that change with tus (Pinton et al,1998).ThisCa2 þ -sensitive photo protein, barrier impairment, such as K þ (Mauro et al, 1998), did not GoAEQmut, is sorted selectively to the Golgi apparatus (Brini change in the HHD epidermis (Fig 4B), con¢rming that the VOL. 121, NO. 4 OCTOBER 2003 ATP2C1 CONTROLS GOLGI CA2 þ 691

Figure1. (A) Localization of the ATP2C1 to the Golgi in subcellular membrane fractions and (B) the ATP2C1 localizes to the Golgi in normal human keratinocytes. (A) Normal keratinocytes were homogenized, the nuclear pellet removed by a low-speed spin, and the remaining membrane fractions were collected by a high-speed spin, resuspended, and layered over a discontinuous sucrose gradient (15%^50% sucrose) for separation by centrifugation. Each fraction was removed and analyzed for colocalization of markers of subcellular membranes (panels 1^4) with ATP2C1 (panel 5)or ATP2A2 (panel 6) by western blot analysis. CM refers to the crude membrane preparation layered onto the sucrose gradient. The Golgi calcium pump ATP2C1 (panel 5) is found mostly in the trans-Golgi fractions, whereas the ER calcium pump ATP2A2 is found in the ER fractions (panel 6). (B) Normal human keratinocytes were immunostained with the trans-Golgi membrane-associated protein p230 or the cis-Golgi matrix protein GM130 (top panels; both shown in red) or immunostained with the ATP2C1 antibody (middle panels; green). Colocalization also is shown by overlapping images (bottom panels) in which colocalization is denoted by yellow. Negative controls omitting the primary antibody demonstrated equal to background nonspeci¢c staining (not shown). In images scanned in the standard (x^y) direction, colocalization in a perinuclear distribution is evident for ATP2C1 and p230, and a z-line scan through cells selected from this image reveals the Golgi-typical nuclear capping in a bipolar fashion. The cis-Golgi matrix protein GM130 localizes to a more restricted perinuclear position and does not completely overlap with ATP2C1 localization. Bar for x^y images, 20 mm; in z-line scan, 5 mm. decrease in Ca2 þ seen in HHD epidermis is independent of DISCUSSION changes in epidermal permeability barrier integrity. These studies demonstrate that ATP2C1-controlled Ca2 þ forms HHD, or benign chronic familial pemphigus, is an autosomal a signi¢cant store of Ca2 þ in keratinocytes and epidermis. dominant blistering skin disease caused by mutations in the PIXE studies further suggest that the ATP2C1 controls ATP2C1 (Hu et al, 2000; Sudbrak et al, 2000). Although the cen- proportionally more Ca2 þ as keratinocytes di¡erentiate in the tral role of the PMR1 and Golgi calcium stores is well established upper epidermis and that active mechanisms, such as the in yeast (Rudolph et al, 1989; Antebi and Fink, 1992; Sorin et al, ATP2C1, participate in the formation of the epidermal calcium 1997; Durr et al, 1998), the ¢nding that mutations of this Ca2 þ gradient (Menon et al, 1985). ATPase produced HHD was the ¢rst to demonstrate that it was 692 BEHNE ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 2. The ATP2C1 protein is decreased in HHD keratinocytes, regardless of the state of cell di¡erentiation. Con£uent normal human and HHD keratinocytes were probed for relative levels of ATP2C1 protein using the ATP2C1 antibody (Santa Cruz Biotechnology Inc.) and western blotting. (A) Keratinocytes taken from one patient (HHD) and a matched normal control (cultured adult human keratinocytes) were cultured in 1.2 mM Ca2 þ, a concentration that induces di¡erentiation. Western blotting revealed decreased ATP2C1 expression in di¡erentiated HHD keratinocytes, compared to normal controls. The size of the ATP2C1 protein did not change in HHD keratinocytes. The size of the human ATP2C1 protein in these blots was approximately 115 kDa. (B) Keratinocytes taken from a di¡erent patient (HHD) and a matched normal control (cultured adult human keratinocytes) were cultured in 0.06 mM Ca2 þ, a concentration that induces proliferation, or 1.2 mM Ca2 þ. Decreased expression of ATP2C1 protein was seen in both undi¡erentiated and di¡erentiated HHD kerati- Figure 3. Golgi Ca2 þ concentrations are decreased in HHD keratinocytes, compared to normal controls. An equal amount of protein was nocytes. Normal and HHD keratinocytes were plated on glass coverslips added to each lane (see Materials and Methods), con¢rmed by reprobing until 60%^80% con£uent and then transfected with the speci¢c Golgi ae- with an antibody to b-actin (C). Densitometry, normalized to b-actin, re- quorin probe GoAEQmut 24 to 48 h before experiments. Keratinocyte vealed reductions in ATP2C1 expression compared to normal human Ca2 þ stores were depleted by treatment with ionomycin. The cells then keratinocytes, as depicted in the bar graph (D). ATP2C1 expression is were incubated with coelenterazine N, and Golgi Ca2 þ stores were re¢lled normalized at 100% for normal human keratinocytes, and ATP2C1 ex- by exposure to extracellular Ca2 þ (see Materials and Methods). Normal pression in HHD keratinocytes is expressed relative to normal human keratinocytes (A) re¢lled more quickly and reached a higher baseline level keratinocytes. than did HHD keratinocytes (B). Each tracing represents data from one experiment. Average values of eight experiments (normal keratinocytes) and nine experiments (HHD keratinocytes) are given under Results. important in other organisms (Hu et al,2000).Inthisstudy,we ¢nd that the human ATP2C1 not only localizes to the Golgi but also controls a signi¢cant Ca2 þ store. In the original description such as defective processing of the ATP2C1 protein or domi- of ATP2C1 mutations, more than half of the identi¢ed mutations nant-negative mechanisms, also may play a role in decreasing predicted prematurely truncated products through frameshifts ATP2C1 protein levels. or a single-base-pair substitution (Hu et al, 2000), suggesting Our previous studies of HHD keratinocytes revealed that a haploinsu⁄ciency pathogenesis. We ¢nd that protein levels of resting cytoplasmic Ca2 þ concentrations were abnormally high, the ATP2C1 are decreased in HHD keratinocytes, consistent with whereas total Ca2 þ stores were abnormally low (Hu et al,2000), a haploinsu⁄ciency pathogenesis, although other mechanisms, consistent with decreased intracellular Ca2 þ sequestration. In VOL. 121, NO. 4 OCTOBER 2003 ATP2C1 CONTROLS GOLGI CA2 þ 693

decreasing intra-Golgi Mn2 þ, could interfere with normal cell- to-cell adhesion by increasing Mn2 þ toxicity (Ton et al,2002). Moreover, Golgi Ca2 þ may be an important and previously unrecognized component of keratinocyte Ca2 þ signaling. Intra- cellular Ca2 þ release in response to raised extracellular Ca2 þ is blunted in HHD keratinocytes (Hu et al, 2000), even though the HHD ER Ca2 þ stores do not di¡er from those of normal keratinocytes (Hu et al, 2000). Decreased Ca2 þ release in HHD keratinocytes is not due to decreases in the Ca2 þ receptor, which is required for normal Ca2 þ signaling in response to raised extracellular Ca2 þ (Tu et al, 2001), because mRNA levels and alterna- tive splicing of the plasma membrane Ca2 þ receptor are normal in HHD keratinocytes (data not shown). These ¢ndings suggest that Golgi Ca2 þ mobilization is essential to stimulate normal Ca2 þ signaling in keratinocytes. 2 þ The identi¢cation of IP3 -sensitive Ca stores controlled by the Golgi ATP2C1 in C. elegans supports this hypothesis (Van Baelen et al, 2001), as does the recent demonstration that a PMR1-controlled Golgi Ca2 þ store produces Ca2 þ oscillations in PMR-1 transfected COS-1 cells (Missiaen et al, 2001). Because extracellular Ca2 þ signals essential processes such as keratinocyte di¡erentiation (Hennings et al, 1983; Pillai et al, 1990; Presland et al, 1995), adhesion (Vasioukhin et al, 2000), motility (Fang et al,1998), and lipid secretion (Lee et al, 1994), blunting cellular responses to extracellular Ca2 þ may have profound e¡ects on keratinocyte and epidermal biology. PIXE measurements of HHD epidermis localize the decrease in epidermal Ca2 þ to the upper epidermis, whereas the lower epidermis and dermis do not show di¡erences in total Ca2 þ. These results correlate well with the clinical, functional impairment seen in HHD, because the acantholysis that Figure 4. ATP2C1 controls a signi¢cant Ca2 þ store in the epidermis. characterizes HHD occurs only in suprabasal cells, where the Biopsies of normal truncal skin from normal (¢lled circles) and HHD (open Ca2 þ values begin to diverge. In addition, these data also suggest squares) volunteers were obtained and analyzed according to Materials and that active mechanisms, such as the ATP2C1, constitute much of Methods. Samples were analyzed with PIXE for concentrations in ppm/dry the epidermal Ca2 þ gradient, which is central to the develop- weight of skin. Samples were oriented so that the skin surface (stratum cor- ment and repair of the epidermal permeability barrier (Menon neum) is labeled as 0 mm. Each sample was measured in triplicate and the et al, 1985; Mauro et al,1998). result presented as the mean7SD. (A)Ca2 þ concentrations in dermis and Mutations in the Golgi Ca2 þ ATPase ATP2C1 cause acantho- epidermis. (B)Kþ concentrations in dermis and epidermis. lysis (HHD), whereas mutations in the ER Ca2 þ ATPase ATP2A2 cause both acantholysis and apoptosis (Darier’s disease), possibly mediated by stress responses caused by the accumulation of unfolded proteins within the ER (reviewed in Paschen 2001) normal keratinocytes, raised extracellular Ca2 þ signals an in- or generation of the proapoptotic molecule caspase 12 (Nakagawa crease in intracellular Ca2 þ by a mechanism that requires both et al, 2000). Thus, the clinical phenotype of acantholysis plus intracellular Ca2 þ release (Tang and Ziboh, 1991; Pillai and Bikle, apoptosis in Darier’s disease likely re£ects the varied functions 1992; Oda et al,2000)andCa2 þ in£ux (Kruszewski et al,1991; of the keratinocyte ATP2C1 versus ATP2A2-controlled Ca2 þ Mauro et al, 1998; Csernoch et al, 2000). Increased Ca2 þ stimulates stores. actin reorganization and ¢lopodia formation that, together with ATP2C1 also is expressed in other tissues, such as liver (Hu et 2 þ E-cadherin complexes, form an ‘‘adhesion zipper’’ (Vasioukhin et al, 2000), and IP3 also induces Ca release from rat liver Golgi al, 2000). Both E-cadherin processing (Hakuno et al,2000)and vesicles (Surroca and Wol¡, 2000). Although HHD generally is desmosome formation (Hashimoto et al, 1995) are disturbed in restricted to the skin, a recent report of HHD with fatal liver in- HHD. jury (Amagai et al, 2001) suggests that this transporter may be im- Several pathologic mechanisms may link the observed defects portant in other organ systems as well. in Golgi Ca2 þ sequestration with the clinical ¢ndings of impaired keratinocyte cell-to-cell adhesion in HHD. HHD kerati- 2 þ nocytes demonstrate abnormally high cytoplasmic Ca and a This work was supported by NIH AR44341 (T.M.M.) and the San Francisco Ve- 2 þ decrease in the cytoplasmic Ca response to stimuli (Hu et al, teran’s A¡airs Hospital and was partially performed under the auspices of the U.S. 2000), and these defects might induce changes in gene expression, Department of Energy at the University of California Lawrence Livermore National or activate protein kinase C, thereby phosphorylating desmopla- Laboratory,under ContractW-7405-Eng-48. kin and disrupting desmosomes (Chakravarthy et al, 1995). Alter- natively, Golgi intraorganelle Ca2 þ depletion might impair post- translational modi¢cation of proteins essential for cell-to-cell adhesion (Amagai et al, 1996). 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