Protein aggregation of SERCA2 mutants associatedwith Darier disease elicits ER stress and apoptosisin keratinocytes

Yin Wang1,*, Allen T. Bruce2, Caixia Tu4, Keli Ma1, Li Zeng1, Pan Zheng1,3, Yang Liu1 and Yan Liu1,*1Department of Surgery, Division of Immunotherapy, Section of General Surgery, University of Michigan Medical Center, Ann Arbor, MI 48109, USA2Department of Dermatology, Division of Immunotherapy, Section of General Surgery, University of Michigan Medical Center, Ann Arbor, MI 48109, USA3Department of Pathology, Division of Immunotherapy, Section of General Surgery, University of Michigan Medical Center, Ann Arbor, MI 48109, USA4Department of Dermatology, The First Affiliated Hospital, Dalian Medical University, Dalian, China

*Authors for correspondence (liuyan@umich.edu; wangyin@umich.edu)

Accepted 16 June 2011Journal of Cell Science 124, 3568–3580� 2011. Published by The Company of Biologists Ltddoi: 10.1242/jcs.084053

SummaryMutations in sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) underlie Darier disease (DD), a dominantly inherited

skin disorder characterized by loss of keratinocyte adhesion (acantholysis) and abnormal keratinization (dyskeratosis) resulting incharacteristic mucocutaneous abnormalities. However, the molecular pathogenic mechanism by which these changes influencekeratinocyte adhesion and viability remains unknown. We show here that SERCA2 protein is extremely sensitive to endoplasmic

reticulum (ER) stress, which typically results in aggregation and insolubility of the protein. Depletion of ER calcium stores is notnecessary for the aggregation but accelerates the progression. Systematic analysis of diverse mutants identical to those found in DDpatients demonstrated that the ER stress initiator is the SERCA2 mutant protein itself. These SERCA2 proteins were found to be less

soluble, to aggregate and to be more polyubiquitinylated. After transduction into primary human epidermal keratinocytes, mutantSERCA2 aggregates elicited ER stress, caused increased numbers of cells to round up and detach from the culture plate, and inducedapoptosis. These mutant induced events were exaggerated by increased ER stress. Furthermore, knockdown SERCA2 in keratinocytesrendered the cells resistant to apoptosis induction. These features of SERCA2 and its mutants establish a mechanistic base to further

elucidate the molecular pathogenesis underlying acantholysis and dyskeratosis in DD.

Key words: SERCA2 aggregation, ER stress, apoptosis, Darier disease, keratinocyte

IntroductionDarier disease (DD) is a rare, autosomal, dominantly inherited

genodermatosis with complete penetrance and variable

expressivity (Godic, 2004; Ringpfeil et al., 2001). Although

there is variable expressivity, affected individuals classically

develop red-brown hyperkeratotic papules characteristically

located on the trunk, scalp, face and neck. Characteristic nail

abnormalities, hand and foot lesions and oral white papules are

often found in affected individuals. Lesions typically first appear

between the ages of 6 and 20 years, and are characterized by

wart-like blemishes on body areas with more exposure to heat,

humidity and sunlight. Patients often experience itchiness and are

troubled by malodors due to frequent skin-lesional infections

with bacteria, yeast and dermatophytic fungi (Ringpfeil et al.,

2001). Mutations in sarcoplasmic/endoplasmic reticulum

calcium ATPase 2 (SERCA2) (Sakuntabhai et al., 1999), a

transmembrane protein located in the endoplasmic reticulum

(ER), which functions in pumping cytosolic calcium into the ER

lumen, have been shown to cause DD. There have been reports

that people with DD may have neuropsychiatric disorders such as

epilepsy, depression and schizoaffective disorders. The

neuropsychiatric issues might be explained in part by non-

genetic factors, including social isolation attributable to the skin

disease (Burge, 1994; Jacobsen et al., 1999; Ruiz-Perez et al.,

1999). The fact that SERCA2b is also highly expressed in

neurons (Baba-Aissa et al., 1998a) suggests that neuron-related

defects might have a role in this disease.

The pathological hallmarks of DD are the loss of desmosomal

adhesion (acantholysis) and abnormal keratinization

(dyskeratosis). The molecular mechanism by which SERCA2

mutations cause these abnormalities is not known, although

previous studies suggest that a depleted ER calcium store might

influence the stability and cell surface expression of desmosomal

proteins (Muller et al., 2006). DD mutations are the result of

missense, nonsense, deletion, insertion and altered splicing

variants of the SERCA2 gene. Because few mutant proteins

were detected in the soluble portion of Triton X-100 cellular

extracts (Ahn et al., 2003), it has been suggested that the mutants

were unstable and could be degraded by the proteasome system.

Although haploinsufficiency of SERCA2 was proposed to

explain the dominant inheritance of DD, the possibility that the

genetic defect is caused by gain-of-function mutation has not

been tested.

The SERCA pumps in humans are encoded by three genes,

ATP2A1, ATP2A2 and ATP2A3. By alternative splicing of their

pre-mRNA, the three genes encode up to a total of 10 isoforms

that possibly are expressed in a tissue-specific manner (Dode

et al., 2003). ATP2A2 encodes three isoforms, SERCA2a,

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SERCA2b and SERCA2c. SERCA2a is expressed in cardiac

muscle (Aubier and Viires, 1998). Relative to SERCA2a,

SERCA2b has an extra C-terminal extension and is

ubiquitously expressed in non-muscle tissues including skin

keratinocytes, cerebellar Purkinjie neurons and hippocampal

pyramidal cells (Baba-Aissa et al., 1998a; Baba-Aissa et al.,

1998b). SERCA2c is missing exon 7 and is expressed in a pattern

similar to SERCA2a (Dally et al., 2006). Compared with

SERCA2a, SERCA2b has a twofold higher affinity for Ca2+

and a 50% lower turnover rate for Ca2+ uptake (Lytton et al.,

1992; Verboomen et al., 1994). Importantly, SERCA2b

expression is induced by diverse ER stressors (Cardozo et al.,

2005; Caspersen et al., 2000), suggesting it is responsible for

Ca2+ uptake under conditions of cellular stress.

Our results show that SERCA2 mutant proteins were not

degraded by proteasomes. Non-degraded mutants formed

Fig. 1. ER stress induces SERCA2 aggregation and apoptosis. (A,B) ER stressors induce the death of keratinocyte HaCaT cells as SERCA2 protein

aggregation increases. The HaCaT cells cultured in a 24-well plate were treated with 1 mM thapsigargin or 1 mg/ml tunicamycin, for the indicated times in DMEM

medium containing 2% FBS. At the end of treatment, 12 wells of cells were lysed for extracting the fraction soluble in 1% Triton X-100 and then the remaining

insoluble fraction was solubilized with 1% SDS plus sonication. The SERCA2 and GRP78 proteins were resolved by 10% SDS-PAGE loaded with reduced, non-

heated samples and detected by western blotting of each soluble fraction (A). The S6K (p70 S6 ribosomal kinase) protein was used as a loading control. The other

12 wells of the cells grown on coverslips were fixed with methanol and stained using Alexa-Fluor-488-linked SERCA2 antibody (B). (C) ER stress induces

keratinocyte apoptosis. The cells were treated with 1 mM thapsigargin or vehicle control for 12 hours. Cell apoptosis and death were detected by flow cytometry

after the cells were stained with FITC–annexin V (for apoptosis) and the nuclear dye DAPI (for death).

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insoluble aggregates that were located in aggresomes. Theseaggregates of mutants caused ER stress and apoptosis and certainmutants exacerbated both responses when mutant-expressingcells were exposed to a second ER stressor. By contrast,

knockdown SERCA2 increased cell insensitivity to apoptosisinduction, a phenomena of dyskeratosis in DD patients. Theseobservations provide a novel interpretation for the acantholytic

and dyskeratotic pathogenesis in DD.

ResultsER stress induces SERCA2 aggregation and apoptosis inhuman primary epidermal keratinocyte HaCaT

Western blot analysis of SERCA2 revealed two bands, identified asa monomer and an oligomer when resolved by SDS-PAGE loaded

with reduced, unheated samples (Fig. 1A). These two bands wereroutinely seen in western blot analysis of SERCA2, as previouslyreported (Lytton et al., 1992). The oligomer of SERCA2 can be

detected by western blot analysis with a longer transfer. However,oligomers of SERCA2 showed decreasing solubility in 1% TritonX-100 lysis solution with increasing exposures to thapsigargin, an

ER stress inducer that is also an inhibitor of SERCA2 (Fig. 1A,right panel). Thapsigargin was more effective than tunicamycin, aN-linked glycosylation inhibitor and promoter of ER stress, at

causing SERCA2 oligomer insolubility (Fig. 1A, right panel). Thefraction of insoluble SERCA2 oligomers that could be dissolved in1% SDS with sonication increased with the time of treatment. Thetwo stressors also increased the expression of the SERCA2

monomers as a function of time (Fig. 1A, left panel), which isconsistent with reported results obtained from rat neuronal cell linePC12 (Caspersen et al., 2000). The ER stress marker GRP78 was

increased in the soluble fraction, demonstrating that both treatmentsinduced ER stress. Staining the thapsigargin-treated cells showedthat approximately 30% of the cells were apoptotic (Fig. 1C).

In immunocytochemical analysis of the immortalized primarykeratinocyte cell line, HaCaT (Fig. 1B), SERCA2 proteins werediffusely distributed in the cytoplasm and appeared to be focused

within the cell boundaries under normal culture conditions(Fig. 1B, 0 hours, no stress). However, 3 hours after stimulation

with thapsigargin SERCA2 proteins were found in small aggregatesor clumps in the cytoplasm or accumulated around the nuclei of the

affected cells (Fig. 1B). At this time point, cells treated withtunicamycin looked moderately affected. Both SERCA2 proteinsand cellular morphology were grossly perturbed by 6 hours of

stimulation with either thapsigargin or tunicamycin (Fig. 1B). Thekeratinocyte layer was largely destroyed by either treatment after

9 hours exposure, with many rounded dying cells detached fromthe culture plate. The sensitivity of the cells to tunicamycin was less

than to thapsigargin, indicating that deficiency in SERCA2 functionenhances SERCA2 aggregative sensitivity to ER stress (Fig. 1B).The breached keratinocyte layer caused by ER stressors resembles

the phenomena observed in the skin lesions of DD patients.However, the question remains as to what is the source of ER stress

and why SERCA2 aggregates in DD.

Missense mutants of SERCA2 gene are insoluble andubiquitinylated, but not degraded by proteasomes

To explore the source of ER stress and SERCA2 aggregation in

DD, we chose to study SERCA2 mutants. SERCA2a andSERCA2b are encoded by the same ATP2A2 gene; SERCA2bis ubiquitously expressed and all SERCA2 mutants identified to

date are of this isoform. Mouse Serca2 has more than 98%identity to human SERCA2 at the amino acid level. To explore

the potential dominant function of ATP2A2 mutations, we usedsite-directed mutagenesis to generate naturally occurring

mutations known to cause skin lesions and neuropsychiatricdisorders, as summarized in Table 1.

Cellular extracts using 1% Triton X-100 lysis buffer have

reduced amounts of SERCA2 mutants compared with the wild-type protein (Ahn et al., 2003). To test the steady state production

and solubility of SERCA2 variants, wild-type (WT) SERCA2 andvarious missense mutants (shown in Fig. 2A) were transientlytransfected into human kidney HEK293 cells instead of HaCaT

Table 1. SERCA2 mutations associated with Darier disease were analyzed in this work

Mutation Exon Function domain Skin features Neuropsychiatric features

MissenseG23E 1 Upstream S1 Less academic achievementN39D 1 Upstream S1 Moderate Hospitalization for violent behaviorL65S 3 M1 FitsG221D 8 b-strand MildC268F 8 M3 HemorrhagicC344Y 8 M4/phosphorylation Mild–moderateI348T 8 Phosphorylation Moderate DepressionF487S 12 Phosphorylation Severe Schizophrenia, seizures, depressionK683E 14 Hinge Mild–moderate Major depressive disorderG749R 15 M5A838P 16 M7 Petit mal epilepsyV843F 17 M7 Depression, epilepsyS920Y 19 M8-M9 loop Moderate–severe EpilepsyH943R 19 M9 Learning difficulties

DeletiondL41 2 S1 Severe Emotional problemsdP42 2 S1 Severe Depression

NonsenseQ790X 16 M6 Multiple neuropsychiatric featuresE917X* 19 M8-–M9 loop

Skin severity is described according to the categorization in previous studies (Ahn et al., 2003; Jacobsen et al., 1999; Ringpfeil et al., 2001; Ruiz-Perez et al.,1999).

* In mouse, the glutamic acid (E) is replaced by the similar glutamine (Q).

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cells, because it is extremely difficult to transfer genes into

keratinocytes. Western blots showed that with the exception of the

L65S, C268F, C344Y and V843F mutants, the mutant SERCA2

proteins appeared insoluble in the 1% Triton X-100 lysis buffer

(Fig. 2B). When comparing heat-denaturated (95 C̊, 3 min)

samples with samples that were not heat treated (Fig. 2B,C), we

found that the heating enhanced the amount of the oligomer of the

WT and the relatively soluble mutants but not the insoluble

mutants, indicating that intrinsic protein misfolding, not any

extrinsic condition, causes the insolubility of SERCA2 mutants.

Overexpression of WT SERCA2 did not elicit protein misfolding

because the ratio of the amount of oligomer to monomer of the WT

proteins was decreased compared with that of the mutants

(Fig. 2C). Anti-FLAG antibody can recognize ectopically

expressed FLAG-tagged SERCA2 as well as an unrelated

protein (non-specific band) shown with the empty vector

transfectant (Fig. 2B). Similar to endogenous SERCA2,

SERCA2–FLAG was resolved as an oligomer and a monomer

by SDS-PAGE loaded with reduced, non-heated samples. The

expression of SERCA2 and its mutants in Neuro2a cells showed a

similar pattern to that in HEK293 cells (data not shown).

Generally, reduced levels of a protein in a Triton X-100

supernatant can result from three processes: nonsense-mediated

mRNA decay (NMD), degradation by proteasomes and formation

of detergent-insoluble aggregates. To measure the proteasome-

mediated degradation of these missense mutants, the transfected

cells were treated with MG132, a commonly used proteasome

inhibitor. Treatment with MG132 did not increase the amount of

the mutant proteins (Fig. 3A), indicating most expressed mutants

were not actively degraded by the proteasome. Partial

degradation occurred in some mutants such as G23E, N39D,

S920Y and H943R; however, blocking degradation with MG132

increased the number of rounded cells expressing mutant proteins

that were aggregated and accumulated around the nuclei

(Fig. 3B). By contrast, 2- to 14-fold more polyubiquitinylated

aggregates were found of the mutant SERCA2 proteins than of

the WT SERCA2 (Fig. 3C). The mutant C268F had the lowest

ratio of polyubiquitinylation of any of the mutants, and this was

also lower than seen for WT SERCA2 (Fig. 3C).

Polyubiquitinylated mutants appeared to accumulate in the

aggresome, supported by colocalization of the FLAG tag with

ubiquitin, proteasome component 20S and vimentin (Fig. 3D).

Fig. 2. Naturally occurring SERCA2 mutant proteins are minimally detected in the Triton-soluble fraction. (A) A diagram of the SERCA2 sequence

depicting the location of the mutants studied in DD patients. (B,C) Ectopic expression of wild-type (WT) and mutant SERCA2. FLAG-tagged mutant and WT

SERCA2 expression plasmids at a concentration of 0.25 mg/per well (B) or 0.1 mg/per well (C) in a 24-well plate were transiently transfected into HEK293 cells.

36 hours (B) or 24 hours (C) after transfection, the cells were lysed with 1% Triton X-100 buffer and the resulting supernatants were resolved by SDS-PAGE

loaded with reduced, heated samples (B) or reduced, non-heated (C) samples. Anti-FLAG antibody was used for the western blots. After being washed, the same

membrane was blotted with anti-b-actin as the loading control.

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We examined the solubility of the mutant proteins and observed

that most tested mutants could not be dissolved in 0.5% Triton X-

100 (Fig. 4A, top panel), but could be partially dissolved in 1%

SDS lysis buffer with sonication (Fig. 4A, bottom panel).

Consistent with their reduced solubility in 1% Triton X-100 lysis

solution (Fig. 2B), the C268F and V843F mutants were also

partially soluble in 0.5% Triton X-100, to different extents; V843F

solubility was less in 0.5% than 1% Triton X-100 buffer (compare

Fig. 4A and Fig. 2B). This demonstrated that some mutants were

expressed at a similar level to the wild-type protein, but had

reduced solubility in Triton X-100 lysis buffer, consistent with

their status as misfolded, ubiquitinylated and aggregated proteins.

Comparison of the solubility of the mutants to their

ubiquitinylation status suggests that misfolding and insolubility

of the mutants were not tightly correlated with ubiquitinylation

status. The reason could be that ubiquitinylated insoluble mutant

proteins were not sufficiently dissolved to be detectable.

To visualize the expression patterns of these mutants, we stained

the transiently transfected HEK293 cells expressing FLAG-tagged

WT or mutant forms of SERCA2. Cells transfected with WT

SERCA2 retained almost normal morphology. By contrast, cells

transfected with most DD-associated SERCA2 mutants assumed a

rounded cell morphology resembling acantholytic keratinocytes,

and contained small clumps or large aggregates of FLAG-tagged

proteins located around the nuclei (Fig. 4B). The cells expressing

the mutants that were only partially soluble in the Triton X-100

buffer (including L65S, C268F and V843F) had an intermediate

phenotype, with more rounded cells expressing the clumped

mutant SERCA2 than WT, but with fewer rounded cells than

for other insoluble mutants (Fig. 4B). These results further

demonstrated that expression of SERCA2 mutants that are

insoluble in Triton X-100 was directly correlated with the

induction of rounded, acantholytic, apoptotic cell morphology

and the formation of intracellular aggregates of

polyubiquitinylated SERCA2. These mutants were poorly

degraded by proteasomes and accumulated in structures of

sequestered, clumped proteins termed aggresomes (Johnston

et al., 1998; Olzmann et al., 2008).

Fig. 3. SERCA2 mutants are polyubiquitinylated and form aggregates in the aggresome. (A,B) Mutants were not degraded by proteasomes. HEK293 cells

were transfected with FLAG-tagged expression plasmids of WT or mutant SERCA2 for 24 hours before treatment or no treatment with 2.5 mM MG132 for an

additional 16 hours. Anti-FLAG antibody was used to detect the expression of WT and mutant proteins by western blotting (A) or immunostaining (B).

(C) Polyubiquitinylation of SERCA2 mutants. After 36 hours of transfection with FLAG-tagged WT or mutant SERCA2 expression plasmids, the cells were lysed

with 1% Triton X-100 and supernatants were immunoprecipitated by anti-FLAG antibody. Anti-ubiquitin blotting detected polyubiquitinylated proteins in

immunoprecipitates. Densitometric quantification of respective polyubiquitin relative to total protein of WT or mutant SERCA2 is shown in the bottom of the

panel. (D) Mutant proteins form aggregates in aggresomes. HEK293 cells were transfected with 0.1 mg/ml FLAG-tagged WT or mutant SERCA2 expression

plasmids for 24 hours. Then the transfected cells were fixed and double stained with antibodies to mouse FLAG and to each of the following proteins: rabbit

ubiquitin (Ub), 20S proteasome, or goat vimentin. Representative results from mutant A838P are shown.

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Deletion and nonsense mutants of SERCA2 are not

degraded and form insoluble aggregates

Of the 122 mutations identified in ATP2A2 in DD patients, 46% are

missense, 12% nonsense, 35% deletion–insertion and 11% alter

mRNA splicing (Godic, 2004). The non-missense and deletion

mutations give rise to truncated proteins. To test if these mutants are

also misfolded, we chose two nonsense mutations and two inframe

deletions with features summarized in Table 1. Deletion of leucine

41 or proline 42 of WT SERCA2, named dL41 and dP42, resulted in

mutant proteins that were mainly distributed perinuclearly. The cells

that expressed these mutants were rounded with perinuclear

aggregates (Fig. 5A). Similar to the deletion mutants, nonsense

mutants of Q790X and E917X accumulated in perinuclear

aggregates (Fig. 5A). Western blots confirmed the expected

molecular mass of these mutants (Fig. 5B). Consistent with the

aggregate formation, these mutants were insoluble in 1% Triton X-

100 lysis buffer although sonication in 1% SDS could partially

dissolve these insoluble aggregates (Fig. 5B). Compared with WT

SERCA2 and deletion mutants, the nonsense mutants were not

completely stable because low amounts of truncated proteins were

detected in the Triton-X-100-insoluble fraction. These data suggest

that similar to the missense mutations, the deletion and nonsense

mutants of SERCA2 generated insoluble truncated proteins that

were also misfolded, aggregated and barely degraded. Possibly,

insoluble, undegradable aggregates of SERCA2 mutants carried by

DD patients could be a common pathogenic factor in DD

development, whether caused by missense, nonsense or deletion

mutations. Evidence to support this assumption is that the effect of

these mutants on endogenous SERCA2 activity is less than 30%

(Fig. 5C). This indicates that there is no apparent correlation

between the altered ER calcium storage and mutant-induced ER

stress; in addition, this shows that deficiency of one SERCA2 allele

is not the leading cause for the development of the dominantly

inherited disease. The measurement of SERCA2 activity is

consistent with a previous report (Ahn et al., 2003). The effect of

these missense mutants on endogenous SERCA2 function is direct

as they were expressed in the ER, although expression of some

mutants in the ER was lower than that of the WT protein (Fig. 5D).

Fig. 4. SERCA2 mutants are insoluble, aggregated and alter cell morphology. (A) Insolubility of SERCA2 mutants. HEK293 cells were sequentially lysed

with 0.5% Triton X-100 (Triton soluble) and 1% SDS with sonication (Triton insoluble) 24 hours after transfection. Anti-FLAG and anti-S6K blots visualized

SERCA2 and the internal control, S6K, proteins, respectively. (B) Aggregation and morphological alteration of cells by SERCA2 mutants. 40 hours after

transfection with FLAG-tagged WT or mutant SERCA2 expression plasmids, the HEK293 cells were fixed with methanol, permeabilized with 0.3% Triton X-100,

and stained with Cy3-conjugated FLAG antibody. Empty vector transfectants showed no staining. Merged images of the Cy3-conjugated FLAG staining and the

DAPI nuclear dye are shown.

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SERCA2 mutants elicit ER stress and induce apoptosis

Cells with insufficiently eliminated aggregates of misfolded

proteins will provoke ER stress and activate the unfolded protein

response (UPR), thereby inducing apoptosis if the UPR is

prolonged and the ER stress is not alleviated (Bernales et al.,

2006; Rutkowski and Kaufman, 2004). ER stress is characterized

by increased levels of ER stress markers including ER resident

chaperon GRP78 and transcriptional factor C/EBP homologous

protein (growth arrest and DNA damage protein 153, also known

as CHOP, GADD153 and DDIT-3) (Jiang and Wek, 2005). We

transiently transfected WT SERCA2 or its missense mutants into

HEK293 cells and measured GRP78 and CHOP levels in the

transfectants after treatment with or without stress stimulator

thapsigargin (Treiman et al., 1998). As shown in Fig. 6A, GRP78

protein levels were significantly increased in many mutant-

expressing cells (except for mutant C268F) compared with WT

SERCA2 cells. Further stressing the transfected cells with

thapsigargin enhanced GRP78 levels to a varied extent

depending on the mutant. Thapsigargin treatment dramatically

increased GRP78 levels in WT and G23E, N39D and C268F

mutant cells that had endured weak ER stress, but did not

increase the already high GRP78 levels in cells expressing the

other SERCA2 mutants (Fig. 6A). HEK293 cells transfected with

the C268F mutant expressed GRP78 and responded to

thapsigargin slightly more than to WT transfections. The

mutant proteins did not elicit detectable expression of the pro-

apoptotic protein, CHOP. However, consistent with the observed

changes in GRP78 levels, CHOP was induced after thapsigargin

treatment in cells transfected with most of the missense SERCA2

mutants. The induction of CHOP was much less in cells

transfected with WT SERCA2 than in cells transfected with all

mutants tested (Fig. 6B). To test whether increased GRP78 and

CHOP levels in response to the mutant SERCA2 proteins

depended on ER calcium depletion by thapsigargin, we used

Fig. 5. Deletion and nonsense mutations of SERCA2 alter cell morphology and form aggregates. (A) Morphologic alteration and aggregate formation of the

indicated SERCA2 mutants. HEK293 cells were transfected with expression plasmids of FLAG-tagged deletion or nonsense mutants for 36 hours and then stained

with Cy3-conjugated FLAG antibody. (B) Insolubility of deletion and nonsense mutants. The same set of transfectants as in A was fractioned with 0.5% Triton X-

100 lysis buffer into supernatant and pellet. The pellet was then washed and dissolved in 1% SDS loading buffer and sonicated. Western blotting detected the

mutant proteins in the two fractions. The internal S6K bands acted as a loading control. (C,D) Activity of SERCA2 mutants in microsomes. HEK293 cells

transfected with expression plasmids of WT or each mutant SERCA2 for 24 hours were subjected to microsome isolation, and SERCA activity was determined as

described in the Materials and Methods. SERCA2 activity is represented as a percentage of endogenous SERCA activity that was arbitrarily set at 100. The

parallel isolated microsomes were directly lysed with 1% Triton X-100 lysis buffer and resolved by a reducing non-heated SDS-PAGE. Western blots using

antibodies to FLAG and GRP78 showed the expression of FLAG-tagged proteins and the ER-resident protein GRP78 (D).

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tunicamycin to stimulate ER stress in the transfected HEK293

cells. Similar to thapsigargin, tunicamycin induced an increase inGRP78 in the cells expressing the mutants G23E, C268F, I348T,

F487S and S920Y; however, tunicamycin increased CHOP to

much higher levels in these mutant-transfected cells than didthapsigargin (Fig. 6C). These results suggest that expression of

the mutants is largely unable to activate UPR at baseline, but

results in full UPR activation in the presence of other ERstressors.

To determine whether SERCA2 mutants induce apoptosis, wemeasured the levels of the apoptotic final executor, cleaved

caspase-3, in mutant-expressing HEK293 cells using western

blotting and immunofluorescence. As seen in Fig. 7, transfectionof most of the mutants resulted in increased levels of activated

caspase-3 compared with the effect of WT SERCA2 (1.5- to 2.5-

fold at baseline), with smaller increases (1.1- to 1.5-fold) seen incells transfected with G23E, C268F, F487S, K683E and V843F.

Those mutant-transfected cells that had higher levels of activated

caspase-3 at baseline exhibited minimal increases in activatedcaspase-3 when treated with thapsigargin (Fig. 7A). However,

the mutant-transfected cells with lower caspase to actin ratios

showed significantly enhanced caspase-3 activity in response tothapsigargin treatment. Consistent with the western blot results,

double immunostaining of transiently transfected cells withantibodies to FLAG tag and active caspase-3 showed that most ofthe rounded-up mutant cells were undergoing apoptosis

(Fig. 7B). The percentage of apoptosis varied for each of themutants but all caused more apoptosis than did transfection withWT SERCA2 (Fig. 7C). Furthermore, the degree to which aSERCA2 mutant promoted apoptosis appeared to correlate with

its insolubility. Activated caspase-3 levels were not correlatedwith increased pre-apoptotic CHOP levels in the mutant-expressing cells. Possibly these SERCA2 mutants induce

apoptosis through mechanisms that are not always dependenton CHOP.

SERCA2 mutants induce ER stress in primary humanepidermal keratinocytes and exacerbate the response toER stressor treatment

To study the effect of SERCA2 mutants in human epidermalkeratinocyte (HEKa) cells, we chose two representative mutants,insoluble G23E and soluble C268F, and transduced them into

Fig. 6. Insoluble SERCA2 mutants elicit ER stress and exacerbate the response to ER stressors. (A–C) The ER stress marker GRP78 is increased in HEK293

cells expressing insoluble mutants. HEK293 cells were transiently transfected with expression plasmids of FLAG-tagged SERCA2 or the indicated mutant

proteins. 24 hours after transfection, the cells were treated with 2 mM thapsigargin for an additional 8 hours (A), 10 hours (B), or with 2 mg/ml tunicamycin for

8 hours (C). Immunoblots show the expression levels of the indicated proteins, and b-actin served as a loading control.

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HEKa cells by lentiviral delivery. After transduction, some cells

expressing mutant proteins appeared to detach from the culture

plate. Immunofluorescence analysis suggested that the mutant

proteins were expressed at higher levels than WT SERCA2, and

resulted in altered morphology (Fig. 8A). Additionally, after

blasticidin selection, the drug-resistant mutants G23E and C268F

also increased the levels of GRP78 and CHOP above that in WT-

SERCA2-transfected cells. The mutant-expressing HEKa cells

were more sensitive to ER stress induction than were HEK293

cells because UPR activation was observed during the static stage

and even in WT-like soluble C268F-infected cells (Fig. 8B).

Tunicamycin treatment enhanced the UPR response in both

mutant- and WT SERCA2-infected cells (Fig. 8B). Flow

cytometry indicated that the mutants G23E and C268F induced

more cell death than did the WT SERCA2 (Fig. 8C). In all three

cases tunicamycin treatment greatly enhanced apoptosis (Fig. 8D).

In either the presence or absence of tunicamycin, the mutant G23E

increased apoptosis compared with WT SERCA2 (Fig. 8D). These

results suggest that SERCA2 mutations might induce apoptosis

and death (acantholysis) of keratinocytes in DD patients.

Knockdown of SERCA2 renders keratinocytes resistant to

apoptosis induction

Because SERCA2 protein is very sensitive to ER stress

stimulation, which results in SERCA2 self aggregation, we

hypothesized that knockdown of SERCA2 protein in

Fig. 7. Insoluble SERCA2 mutants induce apoptosis. (A) Western blot showing that caspase-3 was activated by SERCA2 mutants. HEK293 cells were

transiently transfected with the indicated plasmids for 24 hours and then treated with 2 mM thapsigargin for another 12 hours. The relative ratio of

densitometrically quantified cleaved caspase-3 to b-actin is showed at the bottom, in which the ratio of untreated WT SERCA2 to b-actin was defined as 1.0.

(B) Immunofluorescent staining shows that transfected mutant SERCA2 activated caspase-3. HEK293 cells were transiently transfected with expression plasmids

of WT or mutant SERCA2. After 40 hours, the cells were fixed using methanol, permeabilized with 0.3% Triton X-100 and double stained with mouse Cy3-

conjugated anti-FLAG and rabbit anti-cleaved caspase-3 antibodies followed by anti-rabbit Alexa-Fluor-488 antibody. (C) Percentage of apoptotic cells of

transfectants. The percentage of apoptotic cells were derived from the ratio of cleaved caspase-3-positive cells per 300 FLAG-positive HEK293 cells.

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keratinocytes could elevate the sensitivity threshold for the

apoptosis induction by ER stress. This would explain the

phenomena of dyskeratosis in DD where SERCA2 proteins are

insufficient, either because of the partial or complete degradation

of those mutants by proteasomes, or the aggregation of the WT

SERCA2 protein induced by mutant-elicited ER stress. To test

the hypothesis, we silenced SERCA2 by up to 70–80% in the

keratinocyte line HaCaT. We treated the SERCA2-silenced

[using short hairpin (Sh) RNA] cells with thapsigargin and found

that these cells were resistant to apoptosis induction (Fig. 9B).

This result is consistent to the one showing that heterozygous

Serca2 knockout mice are predisposed to squamous cell

carcinomas (Liu et al., 2001). Although SERCA2 insufficiency

can be compensated for by increase of the Golgi calcium pump

protein ATP2C1 (Foggia et al., 2006), this compensation might

increase the cell insensitivity to ER stress induction. Therefore,

our data show that the aggregation and degradation of mutant

SERCA2 is a cause of acantholysis and dyskeratosis, two

hallmarks of the DD phenotype.

DiscussionDarier disease is caused by mutation of the gene encoding

SERCA2. There is another calcium ATPase residing in the Golgi

apparatus, ATP2C1, which is mutated in Hailey–Hailey (H–H)

disease, a condition that shares histological and sometimes

clinical features with DD. Mutations of the SERCA2 and

ATP2C1 genes lead to a reduction in their calcium pump

activity in vitro (Ahn et al., 2003) and to the moderate depletion

of calcium stores in the keratinocytes of patients with either

disease (Foggia et al., 2006; Hu et al., 2000; Leinonen et al.,

2005). Although haploinsufficiency has been considered to lead

to the development of both diseases, a dominant function of the

mutants has not been tested. Our data show that by exacerbating

UPR, the mutations observed in the DD patients can directly

trigger pathological processes. Although calcium pump activity

deficiency due to SERCA2 mutation correlated with the

sensitivity and severity of SERCA2 aggregation, the causative

mechanism of DD depends on the mutation of SERCA2 itself,

which elicits ER stress possibly to drive the malfunctioning

Fig. 8. Primary human adult epidermal keratinocytes (HEKa) expressing mutant SERCA2 are sensitive to ER stress induction, apoptosis and death.

(A) Expression of WT and mutant SERCA2 in HEKa cells. HEKa cells at approximately 50% confluency were transduced with lentivirus carrying empty vector,

SERCA2, G23E or C268F. The cells were cultured for 4 days after transduction then stained with mouse anti-V5 antibody followed by Texas Red anti-mouse IgG.

(B) SERCA2 mutants induce ER stress. Two days after lentiviral infection, transfected HEKa cells were selected with 6 mg/ml blasticidin and drug-resistant cells

were treated with 2 mg/ml tunicamycin for 8 hours. Total cells, including the detached cells in the culture medium, were collected and lysed for western blotting to

detect ER stress. b-Actin was used as a loading control. (C) SERCA2 mutants enhanced HEKa apoptosis and death induced by tunicamycin. After the same

treatment procedure as in B, cells were collected and cell apoptosis and death were detected by flow cytometry after the cells were stained with FITC-annexin V

(for apoptosis ) and DAPI (for death). (D) Summary of the annexin-V-positive proportion from three separate experiments performed as described in C.

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aggregation of the other WT allele-encoded SERCA2 proteins.

Although we showed that SERCA2-mutation-elicited ER stress

initiates SERCA2 aggregation, other stress factors might play a

role in exacerbating the effects seen in DD. The fact that

heterozygous Serca2 knockout mice do not exhibit any DD

features supports the role of gain-of-function mutations in the

pathogenesis of DD (Liu et al., 2001; Okunade et al., 2007).

Our systematic analysis of the naturally occurring mutations

suggests a general mechanism by which the mutations cause DD.

Almost all the mutant proteins we tested accumulated as

insoluble aggregates that were poorly degraded by proteasomes.

Moreover, the mutant proteins were able to trigger and enhance

the UPR and apoptosis in primary human epidermal

keratinocytes. Our data are consistent with a recent report

showing the upregulation of the ER stress markers GRP78 and

CHOP in DD skin lesions (Onozuka et al., 2006). Surprisingly,

expression of mutant SERCA2 provokes a UPR response that

both stimulates the growth (dyskeratosis) and induces the

apoptosis (acantholysis) of affected keratinocytes. One

explanation is that at an early stage of ER stress there is an

increased expression of GRP78 protein that prevents aggregation

of mutant and wild-type SERCA2 proteins and also elicits an

adaptive response for affected keratinocyte survival. By contrast,

if late stage ER stress occurs because of the presence of the

mutant proteins and/or environmental factors, then CHOP

proteins are induced to promote keratinocyte apoptosis. Owing

to the heterogeneity of keratinocytes in the epidermal basal cell

layer and the sensitivity to ER stress induction, dyskeratosis and

acantholysis are observed simultaneously in the skin lesions of

DD patients.

Another implication of our study is that it might offer a

potential explanation for the neuropsychiatic symptoms reported

in DD patients. Because the mutant proteins are also expressed in

neurons, we speculate that accumulation of the insoluble

aggregates might be an underlying cause of the neuropsychiatic

symptoms. Our data suggest an intriguing correlation between the

insolubility of SERCA2 mutant proteins and neurodisorders

reported in DD patients (Table 1). Even for relatively soluble

mutants such as L65S, C268F and C344Y that weakly elicit ER

stress at static stages in HEK293 cells, mutant-expressing

primary HEKa cells show increased ER stress during static

stages and are vulnerable to apoptosis upon exposure to

additional cellular stresses. For patients with such mutations,

secondary environmental factors could play important roles in the

onset of cutaneous and neurological pathologies. Therefore,

environment factors such as sunlight, infection, sweating and

irradiation exacerbate the skin symptoms of DD. Although

secondary factor effect on the neurological pathology remains to

be tested, it has the simplicity of shared pathogenesis with other

neurodegenerative diseases. Misfolded protein accumulation in

aggresomes due to impairments of proteasome function are

documented causes of neurodegenerative diseases (Olzmann

et al., 2008). Our recent report on the aggregation of laforin

mutant proteins suggests that epilepsy in DD patients might also

be attributable to this mechanism (Liu et al., 2009).

Materials and MethodsAntibodies and reagents

The following antibodies and reagents were used: mouse monoclonal antibodies

specific for SERCA2 (2A7-A1, Abcam; for western blotting), V5 (Invitrogen),ubiquitin (P4D1; Santa Cruz), GRP78/Bip (BD Transduction Laboratories), S6K

(H-9; Santa Cruz), FLAG (M2; Sigma), FLAG-Cy3 (Sigma), b-actin (Sigma) and

CHOP/GADD153 (MA1-250; Thermo Scientific); rabbit antibodies to SERCA2

(NB100-237; Novus), cleaved caspase-3, 20S proteasome (Biomol), polyubiquitin

(Ubi-1; Thermo Scientific), and goat antibody to vimentin (AB1620; Millipore)

for immunofluorescence staining. Thapsigargin, tunicamycin and MG132 were

purchased from Sigma. EpiLife medium (M-EPI-500-CA) and supplement

(HKGS, S-001-5) were bought from GIBCO, Cascade Biologics. The

immortalized human primary epidermal keratinocyte line HaCaT came from Dr.James Varani, Department of Pathology, University of Michigan Medical School.

Plasmids and RT-PCR

The coding region of mouse Serca2b cDNA was amplified by PCR using reverse

transcripts of freshly isolated C57BL/6J mouse brain mRNA as template, and

cloned into a pcDNA vector with a FLAG tag at the N-terminus of Serca2b, namedSERCA2 in the text. After sequencing confirmation of the wild-type Serca2b, all

mutants studied were made by site-directed mutagenesis using Serca2b as a

template, and confirmed by sequencing again. SERCA2, G23E and C268F were

subcloned from their pcDNA plasmids into a lentiviral vector of pLenti6/V5-

TOPO (Invitrogen) after the lentiviral vector was modified. The V5 tag was fused

into the C-terminal sequence of SERCA2 or its mutants. The sequence of the most

effective silencer of SERCA2 used was: Sh2: 59-tactgagatcggcaagatc-39.

Fig. 9. Knockdown of SERCA2 in keratinocytes renders the cells

resistant to apoptosis induction. (A) Efficacy of SERCA2 short hairpin

siRNAs (Sh). HEK293 cells were co-transfected with SERCA2–FLAG

plasmid and the plasmids for scrambled (Sr) or SERCA2 shRNAs for

24 hours. The efficacy was determined by western blot using anti-FLAG

antibody. (B) Silencing SERCA2 increased the resistance of the cells to

apoptosis induction by thapsigargin. HaCaT cells were transduced with

lentivirus carrying SERCA2-Sh or SERCA2-Sr, and selected with 8 mg/ml

blasticidin during 2 weeks in cell culture. The drug-resistant cells that were

more than 95% EGFP positive were treated with 1 mM thapsigargin for

12 hours. Flow cytometry detected the percentage of annexin V (for

apoptosis) and nuclear dye DAPI (for death). Representative flow cytometry

graphs of three separate experiments are shown.

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Transfection and lentivirus preparation

Generally, human embryonic kidney, HEK293, cells were transiently transfectedwith a total of 0.1–0.25 mg plasmid/per well and 0.3–0.75 ml lipofectman-2000/perwell in a 24-well plate for 24–36 hours in Opti-MEM (minimal essential medium)containing 10% fetal bovine serum. To prevent cells from detaching when theculture medium was changed to 0.5 ml Opti-MEM medium, one third of theoriginal culture medium was not removed. Overnight-passaged cells grown toapproximately 75% confluence were used for transfection. Lentiviral vectors ofSERCA2 or its mutants were transfected into HEK293FT cells according to thelentiviral preparation instructions (Invitrogen). The lentivirus was made in a150 mm Falcon culture dish and 50 ml medium was collected and centrifuged at550 g for 10 minutes to remove cell debris. Viral particles obtained aftercentrifugation at 12,857 g overnight were suspended with 2 ml of 10% FBSDMEM and stored at 270 C̊. 50–100 ml of concentrated virus per well of 6-wellplates was used for the transduction in a culture medium containing 6 ml/mlpolybrene. 24 hours after transduction, the infected cells were grown in culture foranother 1–2 days before use.

Western blot and immunoprecipitation

Transfected cells were lysed with pH 7.4 lysis buffer containing 20 mM Tris-HCl,150 mM NaCl, 10 mM NaF, 2 mM dithiothreitol (DTT), 0.5% or 1% Triton X-100, protein and phosphatase inhibitor cocktail (Sigma) at 1:100 dilution.Supernatants of cell lysates were subjected to reducing, heated (95 C̊ for3 minutes) or non-heated 10% SDS-PAGE. Membrane transfer was performedin a semi-dry transfer machine (Bio-Rad) at 100 mA for 3 hours. To allow for hightransfer efficiency of protein aggregates, the transfer was conducted in two transferbuffers: the bottom buffer contained 60 mM Tris-HCl, 40 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 15% methanol, and the top buffer contained60 mM Tris-HCl, 40 mM CAPS, 0.05% SDS. Supernatants of the lysate also wereused for immunoprecipitation (IP) after pre-incubation with protein G beads for2 hours. The cleared supernatant was immunoprecipitated, rotated overnight at4 C̊, then captured by protein G beads with a 4 hour rotation at 4 C̊. Followingintensive washing with 1% Triton X-100 lysis buffer, the IP complex wasdissolved in SDS loading buffer and run in a 10% SDS-PAGE.

Immunofluorescence

Twenty-four hours after transfection, HEK293, HaCaT or HEKa cells were treatedor not with vehicle (DMSO) or 2 mM thapsigargin or tunicamycin for 8 hours andfixed with methanol. The fixed cells were permeabilized with 0.3% Triton X-100,stained with primary mouse antibody to V5 or FLAG, rabbit antibody to SERCA2,ubiquitin or 20S, or goat antibody to vimentin. Secondary antibodies, anti-mouseTexas Red, anti-rabbit Alexa Fluor 488 or anti-goat Alexa Fluor 488 were used fordetection.

Primary human epidermal keratinocyte, adult (HEKa) cultureand transduction

The HEKa cells were from Gibco and cultured in EpiLife medium with supplementsas described by the manufacturer. Second to third passaged HEKa cells werecultured in a 25 cm flask or in a 6-well plate to approximately 70% confluency, andthen transduced with lentivirus for 24 hours. The transduced HEKa cells werecultured in fresh medium for an additional 1–2 days before use in experiments.

Isolation of microsomes

Microsomes (crude ER) were isolated by a serial centrifugation according topreviously described procedures (Li et al., 2004; Parsons et al., 2000). Briefly,HEK293 cells cultured in 6-well plates were transiently transfected with WTSERCA2 or its mutants for 24 hours. After that, the cells were trypsinized andsuspended with 350 ml of low-tonic buffer containing 10 mM Tris-HCl, pH 7.5,0.5 mM MgCl and 1 mM phenylmethanesulfonyl fluoride. The cells werehomogenized with a glass homogenizer for 45 strokes and then made isotonicby the addition of 350 ml buffer containing 0.5 M sucrose, 0.3 M KCl, 6 mM b-mercaptoethanol, 40 mM CaCl2, 10 mM Tris-HCl, pH 7.5. The fractionation wasperformed according to Parsons’s protocol except for the last centrifugation, forwhich 20,000 g for 3 hours was used instead of 100,000 g for 1 hour. The pellets(microsomes) were suspended in 65 ml buffer containing 10 mM Tris-HCl, pH 7.5,0.15 M KCl, 0.25 M sucrose, 20 mM CaCl2 and 3 mM b-mercaptoethanol. TheLowry assay was used for protein determination of 10 ml of the microsomesolution.

Measurement of SERCA2 activity in microsomes

SERCA2 activity was measured using an enzyme-coupled spectrophotometricassay as previously described (Li et al., 2004). The reaction was started by adding25 ml microsomes (equal to 5 mg protein) to 75 ml of assay buffer in the absence(total activity) or presence (activity of thapsigarin-insensitive calcium pumps) of5 mM thapsigargin, and was incubated at 30 C̊ for 60 minutes. The assay buffercontained 25 mM MOPS-KOH, 120 mM KCl, 2 mM MgCl2, 0.45 mM CaCl2,0.5 mM EGTA, 1 mM DTT, 1 mM ATP, 1.5 mM phosphoenolpyruvate, 0.32 mM

NADH, 5 IU/ml pyruvate kinase, 10 IU/ml lactate dehydrogenase, 2 mM calciumionophore A23187 and 5 mM sodium azide, a potent inhibitor of mitochondrialfunction (Solaro and Briggs, 1974). The depletion of NADH was detected byspectrophotometric absorption at 340 nm. The SERCA2 activity was calculated bysubtracting the activity of thapsigargin-insensitive calcium pumps from the totalcalcium pump activity.

AcknowledgementsWe thank Judith Connett for her assistance in revising the English ofthe manuscript.

FundingThis work is supported by the National Institutes of Health [grantnumber 1R21NS062391]. Deposited in PMC for release after 12months.

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