ORAI1 calcium channel orchestrates skin homeostasisMatthieu Vandenberghea,1, Maylis Raphaëla,1, V’yacheslav Lehen’kyia,1, Dmitri Gordienkoa,2, Ryan Hastieb,Thierry Oddosc, Anjana Raob, Patrick G. Hoganb,3, Roman Skrymaa,3, and Natalia Prevarskayaa,3,4

aInstitut National de la Santé et de la Recherche Médicale U1003, Equipe Labellisée par la Ligue Nationale Contre le Cancer, Laboratory of ExcellenceIon Channel Science and Therapeutics, Université des Sciences et Technologies de Lille, 59650 Villeneuve d’Ascq, France; bLa Jolla Institute for Allergyand Immunology, La Jolla, CA 92037; and cPharmacology Department, Johnson and Johnson Santé Beauté France, Campus de Maigremont, 27100 Val deReuil, France

Edited* by Michael J. Berridge, The Babraham Institute, Cambridge, United Kingdom, and approved October 25, 2013 (received for review June 4, 2013)

To achieve and maintain skin architecture and homeostasis,keratinocytes must intricately balance growth, differentiation, andpolarized motility known to be governed by calcium. Orai1 is a poresubunit of a store-operated Ca2+ channel that is a major molecularcounterpart for Ca2+ influx in nonexcitable cells. To elucidate thephysiological significance of Orai1 in skin, we studied its functionsin epidermis of mice, with targeted disruption of the orai1 gene,human skin sections, and primary keratinocytes. We demonstratethat Orai1 protein is mainly confined to the basal layer of epidermiswhere it plays a critical role to control keratinocyte proliferation andpolarized motility. Orai1 loss of function alters keratinocyte differen-tiation both in vitro and in vivo. Exploring underlying mechanisms,we show that the activation of Orai1-mediated calcium entry leads toenhancing focal adhesion turnover via a PKCβ-Calpain-focal adhesionkinase pathway. Our findings provide insight into the functions ofthe Orai1 channel in the maintenance of skin homeostasis.

calcium signaling | cell migration | orai1 KO mice

The involvement of calcium-dependent mechanisms in theinduction and regulation of keratinocyte proliferation, mi-

gration, and differentiation is now well established (1–3). Kera-tinocytes are arranged in highly organized, specialized layersaccording to their functions and the programmed life cycle.Proliferating keratinocytes comprise the stratum basale. Basal-cell proliferation is appreciably higher and inversely correlatedwith the calcium gradient in the skin, reflecting the importanceof calcium signaling in differentiation (3). As a result of pro-liferation, keratinocytes leave the stratum basale, moving towardthe exterior with the onset of differentiation in the stratum spi-nosum. Differentiation is completed in the stratum granulosum,thereby constituting the enucleated stratum corneum, whichplays the major role as a permeability barrier (1). Besides dif-ferentiation and proliferation, the balance of which determinesthe epidermis physiology, the polarized motility of keratinocytesfollows the same vertical pathway, suggesting its crucial impor-tance for skin homeostasis (4).For years, calcium has been considered as a potent inducer of

keratinocyte differentiation; for this reason, calcium channelshave been suggested to be indispensable in its promotion. Of them,store-operated calcium channels (SOCs) are a major mechanismof Ca2+ entry in nonexcitable cells (5–7). A molecular candidatefor SOC termed Orai1 has been identified and characterized (8–12). Numerous studies have demonstrated that Orai1 mediatescalcium release-activated currents and SOC in a large variety ofcells and is involved in a wide range of cell functions, includingendothelial cell proliferation (13), lymphocyte proliferation (14),and mast cell activation (15), as well as skeletal muscle developmentand a contractile function (16). However, the role of Orai1 in skinphysiology remains poorly understood. The phenotypic features ofthe homozygous orai1−/−mice have been recently shown as sporadichair loss, resembling the cyclical alopecia, thinner epidermis withlower cell density, and narrower follicles (17), which indicates theimportant role of the Orai1 channel in skin homeostasis. Althoughthe first findings on the role of Orai1 in differentiation and migra-tion of isolated keratinocytes have very recently appeared (18, 19),they do not reflect the complex role of this channel in the overall

processes of skin homeostasis. In the present study, using bothhuman primary keratinocytes and the keratinocytes obtained fromorai1−/− mice, we found a previously undescribed role of Orai1 inepidermal physiology. Indeed, in contrast to its expected pro-differentiative role, we show that Orai1 constitutively inhibits ter-minal keratinocyte differentiation and is indispensable for thephysiological control of proliferation and migration of basal ker-atinocytes. We demonstrate that Orai1 protein is mainly confinedto the basal layer of the epidermis where it plays a critical role inthe control of keratinocyte proliferation and polarized motility byenhancing focal adhesion turnover via the EGFR-PKCβ-Calpain-focal adhesion kinase (FAK) pathway. Orai1 loss of functiondecreases keratinocyte proliferation and inhibits directional mi-gration, thereby accelerating the expression of differentiation-regulating genes. Finally, Orai1 loss of function alters the skinhomeostasis in an in vivo mice model, confirming our findingsobtained on primary keratinocytes.

ResultsOrai1 Protein Is Mostly Expressed in Stratum Basale and DiminishesDuring Differentiation. Firstly, we have studied the expression ofOrai1 protein in human skin sections (Fig. 1). Immunohisto-chemical studies showed that the Orai1 protein is mostlyexpressed in the stratum basale of human epidermis, with a slightpresence in upper layers of the skin (Fig. 1A). The same ten-dency was observed for the STIM1 protein, which was also

Significance

The epidermis of the skin is composed of keratinocytes that areorganized in several layers. Basal cells divide and produce cellsmoving outwards the epidermis while undergoing the processof terminal differentiation, crucial for the barrier function of theskin. Calcium is an indispensible ion for differentiation, and cal-cium channels are of primary importance. Unexpectedly, wediscovered that the Orai1 calcium channel is mainly expressed inthe basal layer, functioning to negatively control differentiation.The Orai1 channel supplies calcium to sustain proliferation and,in particular, to drive migration of keratinocytes, both processesbeing the feature of basal keratinocytes.

Author contributions: V.L. and N.P. designed research; M.V., M.R., V.L., D.G., and R.H.performed research; M.R., R.H., T.O., A.R., and P.G.H. contributed new reagents/analytictools; M.V., M.R., V.L., T.O., A.R., P.G.H., R.S., and N.P. analyzed data; and V.L., P.G.H., R.S.,and N.P. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Freely available online through the PNAS open access option.1M.V., M.R., and V.L. contributed equally to this work.2Second address: Laboratory of Molecular Pharmacology and Biophysics of Cell Signalling,State Key Laboratory of Molecular and Cell Biology, O. O. Bogomoletz Institute of Phys-iology, Kiev 01024, Ukraine.

3P.G.H., R.S., and N.P. contributed equally to this work.4To whom correspondence should be addressed. E-mail: Natacha.Prevarskaya@univ-lille1.fr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1310394110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1310394110 PNAS | Published online November 25, 2013 | E4839–E4848

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predominantly expressed in basal layers. In contrast to theseproteins, the staining of early differentiation markers like invo-lucrin (IVL) and cytokeratin 10 (KRT10) showed patterns ofexpression with an increasing gradient toward upper layers suchas the stratum spinosum, granulosum. The late differentiationmarker fillagrin (FLG) is mainly expressed in the most superficiallayer of the epidermis: the stratum corneum (Fig. 1A). Thedistribution of STIM1 was found to mirror that of Orai1 and ismostly confined to the basal layer of skin although, according toimmunohistochemical data, the expression of both is still con-served in the upper layers of epidermis. Although several butvarious differentiation markers are shown in each experiment,the expression of all of the differentiation markers, such as early

(e.g., IVL, KRT1 and -10), intermediate (e.g., TGM1), and late(e.g., Filaggrin, Loricrin), was studied.To confirm the immunohistochemical studies, we have studied

the expression of Orai1, STIM1, and the late differentiationmarker FLG in human primary keratinocytes (hPKs) using real-time quantitative PCR (Fig. 1B). Quantification of the mRNAtranscripts from three independent experiments showing theexpression levels of Orai1, STIM1, and FLG in hPK kept in 0.07mM Ca2+ [undifferentiated hPK (UhPK)] or hPK cells treatedwith 1.7 mM Ca2+ during 1, 3, or 6 d [differentiated hPK(DhPK)] is indicated in Fig. 1B. The expression data for Orai1,STIM1, and FLG confirmed those of immunohistochemicalstudies indicating the down-regulation of these proteins duringthe differentiation process in contrast to the FLG, whose ex-pression increases during differentiation.To study the role of Orai1 in calcium-induced differentiation

of hPK, we used a so-called Ca2+-switch protocol. The aim wasto see whether the knockdown of Orai1 protein may influencethe onset of keratinocyte differentiation. Thus, the cells are pre-transfected with the Orai1-specific siRNA tested preliminarily asshown in Fig. S1A. After 24 h, 1.7 mM Ca2+ is added to UhPK totrigger differentiation for an additional 24 h. The control hPKcells kept in 0.07 mM Ca2+ and control hPK cells with the in-duced Ca2+ switch were transfected with siCtrl. The pretrans-fection of hPK cells with siOrai1 significantly induced the ex-pression of differentiation markers such as cytokeratin 1 (KRT1),IVL, keratinocyte transglutaminase (TGM1), and even FLG,which was superior to the extent of Ca2+ switch itself (Fig. 1C).These data strongly indicate the important role of Orai1 in theprocess of keratinocyte differentiation.Because the previous experiment suggested the probable nega-

tive role of Orai1 in differentiation, we decided to see whether theknockdown of Orai1 protein in the low Ca2+ solution (undifferen-tiated, UhPK) is able to trigger differentiation per se (Fig. 1D).The pretransfection of UhPK cells with siOrai1 in 0.07 mM Ca2+

for 24 h has slightly but significantly increased the expression ofearly differentiation marker IVL whereas the expression of latedifferentiation marker FLG remained unchanged, confirming thecrucial role of calcium (superior to 0.07 mM, entering via otherchannels) in differentiation processes. These data suggest thata decrease in Ca2+ entry through the Orai1 channels contributesto the onset and/or progression of differentiation.

Orai1 Is the Main Component of the Store-Operated Current in HumanKeratinocytes. The entry of calcium through the plasma mem-brane into the cytoplasm [Ca2+]i in response to the decrease incalcium content of the endoplasmic reticulum [Ca2+]ER wasstudied (Fig. 2). Depletion of endoplasmic reticulum (ER) storesvia activation of IP3 receptors or by inhibition of the ER SERCApump with thapsigargin (TG) induced a significant store-oper-ated calcium entry (SOCE) in undifferentiated hPK (UhPK)compared with differentiated (DhPK) cells (Fig. 2 A and C, re-spectively). The normalized quantification of this current is givenin Fig. 2E. This entry, induced either by IP3 or TG, was suc-cessfully blocked by inhibition of the Orai1 channel and STIM1protein using the transfection of UhPK with the respective siRNAsagainst these proteins (Fig. 2 B and D). The quantification histo-gram of this SOCE inhibition is shown in Fig. 2F. We can concludethat the Orai1 channel and STIM1 proteins are the main compo-nents of store-operated calcium current in human keratinocytesand that this SOCE is significantly down-regulated following dif-ferentiation of hPK.

Orai1 Channel Is Involved in Proliferation and Migration ofUndifferentiated hPK. Having discovered the expression and therole of the Orai1 channel in SOCE of UhPK corresponding toundifferentiated keratinocytes, we have sought to study its role incell proliferation and migration, the characteristic features ofkeratinocytes derived from stratum basale. The indirect colori-metric cell viability assay (MTS) showed a strong inhibition of

Fig. 1. Orai1 expression in human skin and its role in keratinocytes differ-entiation. (A) immunohistochemical studies for the expression of Orai1,STIM1, IVL, KRT10, and FLG in human skin sections. Top Left shows the re-spective control for our stainings using secondary antibody only. Insets(Right) show enlarged images of the boxed regions. Different layers (Inset,Top Left) in epidermis are: (i) stratum basale; (ii) stratum spinosum; (iii)stratum granulosum; (iv) stratum lucidum; (v) stratum corneum. (Scale bars:50 μm.) (B) Real-time quantitative PCR from three independent experi-ments showing the expression levels of Orai1, STIM1, and FLG in hPK keptin 0.07 mM Ca2+ (UhPK), or hPK cells treated with 1.7 mM Ca2+ during 1, 3, or6 d (DhPK). Data were normalized compared with HPRT gene expression.Data represented are mean ± SEM; *P < 0.05; **P < 0.01, n = 3. (C) Theeffects of Orai1 knockdown on the expression of FLG, IVL, KRT1, and TGM1transcripts induced by calcium switch (1.7 mM Ca2+, 24 h). Data were nor-malized compared with HPRT gene expression. Data represented are mean ±SEM; *P < 0.05; **P < 0.01; ***P < 0.001; n = 3. (D) The expression of IVL andFLG in hPK kept in 0.07 mM Ca2+ induced by Orai1 knockdown for 24 h. Datawere normalized compared with HPRT gene expression. Data representedare mean ± SEM; **P < 0.01; n = 3.

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undifferentiated hPK (UhPK) proliferation by the extinction ofboth Orai1 and STIM1 proteins (Fig. 3A). These data wereconfirmed by the direct cell counting following 48 h of celltransfections with siRNA against Orai1 and STIM1 (Fig. 3B).Further, the issue of cell migration was addressed. We have used

two methods: wound healing and Transwell assays. The down-regulation of Orai1 protein by siRNA has decreased UhPK cellability to migrate and to “heal the wound” (Fig. 3C, Upper). In theother series of experiments, we inhibited FAK with 10 μMFAKi 14inhibitor for the indicated period (Fig. 3C, Lower). This inhibitionarrested the cell migration. To determine whether the intracellularcalcium ([Ca2+]i) may have a role, we blocked the [Ca2+]i with thecell-permeable Ca2+-chelator BAPTA-AM (50 μM). The effect ofchelating [Ca2+]i was also prominent and arrested hPK cell mi-gration in the time scale of 3 d (Fig. 3C,Lower). Thus, Orai1, as wellas [Ca2+]i, is extremely important for hPK cell migration.The use of a Transwell assay enabled us to distinguish pro-

liferating and migrating cells. This assay showed a significant in-hibition of UhPK migration to the lower chamber of the Transwell

system when Orai1 and STIM1 proteins are down-regulated (Fig.3D). The same effects were observed when FAK was inhibitedby FAKi 14, or [Ca2+]i is chelated by BAPTA-AM. In addition,Z-LLY-FMK, a specific inhibitor of calcium-dependent proteasecalpain, a protease well-known to cleave FAK, at 10 μM success-fully blocked hPK migration, confirming its important role in focaladhesion turnover.We have further studied the expression of FAK in human skin

sections and the level of FAK autophosphorylation. The ex-pression of FAK, especially its tyrosin-397 autophosphorylatedform (pFAK397), crucial for its activation and involvement infocal adhesion turnover, was predominantly located in the basallayer as for Orai1 and STIM1 (Fig. 3E). The independent knock-downs of both Orai1 and STIM1 significantly diminished thequantity of autophosphorylated FAK397 protein, compared with thetotal FAK protein (Fig. 3F). The efficiency of transfection, as wellas the quantification of blots, is shown in Fig. 3G. The otherchannels, such as TRPC1 and TRPV6, were also studied and didnot show any significant decrease in FAK397 protein (Fig. S1B).Further, we have checked whether the inhibition of FAK auto-phosphorylation could have a role by inhibiting FAK with its in-hibitor, 10 μM FAKi 14 or by sequestrating [Ca2+]i with the cell-permeable chelator BAPTA-AM. The level of pFAK397 proteinwas significantly attenuated by these agents (Fig. 3H), both of whichdecreased the quantity of pFAK397 protein whereas the total FAKwas unchanged (see quantifications in Fig. S1C). Therefore, STIM1/Orai1-mediated Ca2+ influx is an important controller of FAKautophosphorylation and focal adhesion turnover.Finally, because the inhibitor of calpain, Z-LLY-FMK, suc-

cessfully blocked keratinocyte migration, we have studied therole of Orai1 and STIM1 proteins in calpain activity using thefluorometric calpain activity assay kit from Merck ChemicalsLtd. The activity of endogenous calpains was decreased uponOrai1 and STIM1 knockdowns (Fig. 4C), suggesting the possibleactivation of calcium-dependent calpain proteases downstreamof the Orai1 channel.

Orai1 and STIM1 Regulate Focal Adhesion Turnover and UndifferentiatedhPK Polarized Motility. The reorganization of the cell cytoskeletonis necessary to exercise cell movement in space. As a part ofthis complex process, the formation and disassembly of focal-adhesion contacts is a prerequisite for the dynamics of thisprocess. The turnover of these focal adhesions has been mon-itored using confocal microscopy (Fig. 4 A and B). Two proteinscrucial for cell movement, pFAK397 and paxillin, were stainedtogether with F-actin, a polymerized form of G-actin. The knock-down of Orai1 and STIM1 proteins provoked the aggregation ofpFAK397 protein and inhibited focal adhesion contacts turnover, aswell as stimulated stress-fiber formation compared with a total ex-tinction of focal adhesion contacts when FAK protein was inhibitedby siRNA (Fig. 4A). The knockdown of Orai1 and FAK yieldeda constricted cell morphology and stress-fiber formation (Fig. 4B).The other protein important for focal adhesion contacts formation,paxillin, was also aggregated. These data demonstrate the impor-tant role of Orai1 and STIM1 in focal-adhesion turnover crucial forcell migration.Because the focal adhesion turnover is necessary for kerati-

nocyte migration, we have studied the role of Orai1 and STIM1in polarized motility of undifferentiated hPK (Fig. 5). In controlexperiments using confocal microscopy and hPK staining withCellMask, we have shown that differentiated hPK lack polarizedmotility and cell migration in general (Movies S1 and S2). Theseeffects were mimicked by the use of the FAK inhibitor in UhPK(Fig. 5A and Movie S3). The successful knockdown of Orai1(Movie S4), STIM1 (Movie S5), and FAK (Movie S6) proteins inUhPK completely arrested cell migration of UhPK, implying theimportant role of these proteins in polarized motility of humankeratinocytes (Fig. 5B).

Fig. 2. Store-operated current in human primary keratinocytes. (A and C)Representative time courses of ISOC development (current density, measuredat −100 mV holding potential) in UhPK cells (black traces, kept in 0.07 mMCa2+) and DhPK cells (gray traces, kept in 1.7 mM Ca2+) in response to thedialysis of 100 μM IP3 (A) or 1 μM TG (C). (B and D) Representative timecourses of ISOC development (current density, measured at −100 mV holdingpotential) in UhPK cells (kept in 0.07 mM Ca2+) pretreated with siRNAagainst Orai1 (gray traces), STIM1 (light gray traces), compared with siCtrl(black traces) in response to the dialysis of 100 μM IP3 (B) or 1 μM TG (D). Inthe case of TG-induced SOCE (C and D), the presence of 10 mM BAPTA in thepipette may partially account for the SOCE development. (E) Quantificationof the maximum amplitude of IP3- and TG-induced ISOC densities in UhPKcells (white column, kept in 0.07 mM Ca2+) and DhPK cells (black column,kept in 1.7 mM Ca2+). Data shown are normalized. Mean ± SEM; *P < 0.05;n = 32 for each condition. (F) Quantification of the maximum amplitude ofIP3- and TG-induced ISOC densities in UhPK cells (kept in 0.07 mM Ca2+)pretreated with siRNA against Orai1 (gray columns), STIM1 (black columns),compared with siCtrl (white columns). Data shown are mean ± SEM; *P <0.05; n = 34 for each condition.

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The Role of the EGF-Dependent Pathway and SOCE in KeratinocytePolarized Motility. To have an insight into the possible mecha-nisms leading to migration/polarized motility via the Orai1channel, we have investigated the possible role of endothelialgrowth factor (EGF) signaling in activation of hPK cell migra-tion. Our data show that EGF stimulates the migration in thewound-healing assay whereas siOrai1 pretreatment of cellsinhibits these effects (Fig. 6A). The same protocol has beenperformed in Transwell assays, and the same data have beenobtained (Fig. 6B), suggesting the pivotal role of EGF in kera-tinocyte migration and the important contribution of Orai1channels in this pathway. To prove the role of Orai1 channelsand SOCE in this signaling, a panel of pharmacological inhibitorswas used in the study. The migration studies using Transwell assayshave been preformed using the pharmacological inhibitors againstcalpain I (iCalpI, 10 nM), calpain II (iCalpII, 1 μM), Orai1 (BTP2,1 μM), STIM1 (ML9, 10 μM), and PKCβ (iPKCβ, 100 nM). All ofthem, except for the inhibitor of calpain II protease, diminished therate of keratinocyte migration (Fig. 6C). Moreover, the otherinhibitors of SOCE, such as 2-APB, La3+, and Gd3+, and the veryrecently appeared inhibitors of Orai1 selectivity filter, GSK-7975Aand GSK-5503A (20), were used and showed the significant de-crease in hPK cell migration (Fig. S1D). Further, because theautophosphorylation of FAK at the tyrosine 397 residue is crucialfor its activation and involvement in focal adhesion turnover, we

further studied the effects of pharmacological inhibition of PKCand SOCE pathway on FAK397 autophosphorylation. Indeed,the inhibition of Orai1 (BTP2, 1 μM), calcium-dependent PKC(Gö6976, 1 μM), and PLC (U73122, 1 μM) decreased the level ofFAK397 autophosphorylation induced by EGF (Fig. 6D). As for themigration, the other inhibitors of SOCE, such as 2-APB, La3+, andthe very recently appeared inhibitors of Orai1 selectivity filter,GSK-7975A and GSK-5503A, were tested and showed a decreasein pFAK397 level (Fig. S1E). EGF also induced the cleavage ofFAK, yielding an 80-kDa fragment, the result of FAK degradationby proteases such as calpain and therefore enhanced its turnover(Fig. 6E). The effects of pharmacological inhibition of FAK deg-radation under the inhibition of Orai1 (BTP2, 1 μM), STIM1(ML9, 10 μM), two PKC inhibitors (iPKCβ, 0.1 μM and Gö6976,1 μM), and PLCγ (U73122, 1 μM) are shown in Fig. 6E. Moreover,the quantity of 80-kDa fragment of the cleaved FAK was dimin-ished following both siOrai1 and siSTIM1 treatments, but notthose of siTRPC1 and siTRPV6 (Fig. S1B). The expression ofEGFR and paxillin was also monitored as a control that the ob-served effects are solely due to the intracellular messengers down-stream to the EGFR, and the number of structural proteins such aspaxillin does not change during the treatments (Fig. S1F).Finally, EGF by itself was capable of inducing SOCE, and this

SOCE induced by EGF was successfully inhibited by siRNAagainst Orai1, suggesting the direct role of this channel in EGF-

Fig. 3. The role of Orai1 and STIM1 in proliferation and mi-gration of human keratinocytes. (A) A time course of UhPK cell(kept in 0.07 mM Ca2+) growth revealed by indirect colorimetricMTS assay reflecting the number of viable cells. Cells weretransfected with siRNA against Orai1 (gray squares), STIM1(black triangles), compared with siCtrl (white circles). Datarepresented are mean ± SEM; *P < 0.05; **P < 0.01, n = 3. (B)Relative cell number of UhPK (kept in 0.07 mM Ca2+) cells after48 h of transfection with siRNA against Orai1 (gray column),STIM1 (black column), compared with siCtrl (white column).Data represented are mean ± SEM; **P < 0.01, n = 3. (C) Arepresentative experiment of three independent experimentsof wound-healing assay of UhPK (kept in 0.07 mM Ca2+) cellstransfected for 24 h with siRNA against Orai1 versus siCtrl(Upper), and UhPK cells treated with DMSO, 10 μM FAKi 14,and 50 μM BAPTA-AM for the indicated period (Lower). Pic-tures were taken every 24 h during 3 d. (D) Cell migration assayusing Transwell chambers of UhPK (kept in 0.07 mM Ca2+) cellstransfected with 60 nM of siRNA against Orai1, STIM1 versussiCtrl as well as FAKi 14, BAPTA-AM, and Z-LLY-FMK (10 μM)versus DMSO. Cells were allowed to migrate for 24 h from thetime of transfection. Data shown are mean ± SEM; *P < 0.05;**P < 0.01, n = 3 done in quadruplicate. (E) The immunos-taining of phosphorylated FAK397 protein in human skin sec-tions. The small black box corresponds to the magnified skinarea. (Scale bar: 50 μm.) (F) A representative immunoblottingshowing the level of autophosphorylation of FAK397 comparedwith overall FAK in UhPK cells treated with 60 nM siRNAagainst siOrai1 and siSTIM1 for 24 h. siFAK has been used asa positive control. (G) A quantification histogram of FAK397

autophosphorylation in UhPK cells (of a representative immu-noblotting shown in F). Data represented are mean ± SEM;**P < 0.01, n = 3. (H) The autophosphorylation of FAK397 inUhPK cells treated with FAKi 14 and BAPTA-AM; DMSO wasused as a control.

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induced SOCE (Fig. 6F). Further, the PLC inhibitor U73122 (10μM, 30 min) completely inhibited ER Ca2+ release induced byEGF (Fig. 6G). In an additional series of experiments, weinhibited the expression of FAK with its respective siRNA, andwe observed no difference in SOC current, suggesting that FAKsignaling is definitely downstream of Orai1 signaling (Fig. S1G).

Keratinocyte Physiology and Epidermis Barrier Are Impaired in orai1−/−

Mice. To confirm the importance of our findings obtained in vitro,we exploited the recently created orai1−/− mouse model (17).SOCE induced by 1 μM TG is smaller in keratinocytes fromorai1−/− mice than in control ones from orai1+/+ or orai1+/− (Fig.7A). The proliferation rate is remarkably affected (Fig. 7 B and C).The ability of keratinocytes to migrate was assessed using theTranswell assay and showed a significant decrease in the numberof migrating cells obtained from orai1−/− mice compared with

control ones from orai1+/+ mice irrespective of whether EGF wasused or not (Fig. 7D). We have also studied the epidermis inorai1−/− mice compared with orai1+/+ mice, using immunostainingprocedures and confocal microscopy (Fig. 7E). Immunostainingusing anti-KRT5, KRT10, and loricrin (LOR) in orai1−/− miceshowed the impaired expression of differentiation markers suchthat the marker of basal cell KRT5, the marker of early differ-entiation KRT10, and the late differentiation LOR, are equallyexpressed in all layers (Fig. 7E). Conversely, the epidermis oforai1+/+mice had the well-distinguished classical expression of KRT5in basal and suprabasal layer, the increasing expression of KRT10above the suprabasal layer, and the expression of LOR in theterminal cornified layer. We also observed the disintegration anddetaching of the stratum corneum layer in orai1−/− mice, witha significant decrease in both skin and epidermis thickness.

DiscussionIn the current study, we report several major findings: (i) wehave identified in human epidermis an Orai1 protein whose ex-pression is predominantly confined to the basal layer of epider-mis; (ii) the Orai1 channel is a principal source of endogenousSOCE in human keratinocytes; (iii) Orai1 is involved in pro-liferation and migration, and keratinocyte differentiation is un-der the negative control of the Orai1 channel; and (iv) the Orai1channel mediates polarized motility in keratinocytes both basaland induced by EGF by supplying calcium via SOCE used to (i)activate PKCβ, which enhances focal adhesion contact formationvia stimulation of pFAK397 autophosphorylation and associationof pFAK397 and paxillin with actin filaments, and (ii) activate acalcium-dependent protease calpain participating in FAK deg-radation and therefore accelerating FAK turnover, both of thempromoting polarized cell migration. Finally, the keratinocytesfrom Orai1 KO mice exhibit remarkably decreased migration,

Fig. 4. Involvement of Orai1 and STIM1 in focal adhesion turnover.Immunostaining of F-actin (red) and pFAK (green, A) or paxillin (green, B) ofUhPK cells (kept in 0.07 mM Ca2+). UhPK cells were transfected with siOrai1,siSTIM1, siFAK, and siCtrl for 24 h before fixation. Small white boxes corre-spond to the magnified area of the image where pFAK (A) or paxillin (B) areshown in gray scale. (Scale bars: 10 μm.) (C) Calpain activity in UhPK cellstreated with 60 nM siRNA against siOrai1 and siSTIM1 for 24 h. Cell lysateswere obtained and assayed according to the fluorometric calpain activity kit(Merck Chemicals Ltd.) and compared with a provided positive control(Calpain).

Fig. 5. Polarized motility of human keratinocytes affected by Orai1 andSTIM1. The motility of hPK cells (kept in 0.07 mM Ca2+) was monitoredduring 1 h using confocal microscopy imaging of the hPK cells stained withCellMask for 30 min. (A) Polarized motility of UhPK cells (kept in 0.07 mMCa2+) and DhPK cells (kept in 1.7 mM Ca2+) compared with UhPK cells pre-treated with 10 μM FAK inhibitor 14 for 30 min. (B) Polarized motility ofUhPK cells (kept in 0.07 mM Ca2+) transfected with siRNA against Orai1,STIM1, and FAK versus siCtrl.

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proliferation, and impaired differentiation, yielding impairedepidermis formation.In the present study, we have shown that Orai1 is one of the

most important pore-forming entities in the plasma membraneand, together with STIM1 as a signal transducer from the ER,represents the major molecular component of SOCE in humanprimary keratinocytes: siRNA-mediated knockout of any of themstrongly diminishes ISOC in keratinocytes. The translocation ofSTIM1 to the keratinocyte plasma membrane following agoniststimulation was shown for HaCaT cells (21); however, the mo-lecular mechanisms and the role of Orai1/STIM1 couplingremained obscure. Although the role of Orai1/STIM1-mediatedSOCE in the HaCaT keratinocyte cell line has recently beendescribed (18), it does not address the physiological role of thischannel in skin. In fact, the HaCaT cell line is an artificial cellline of spontaneously immortalized keratinocytes (22). This an-euploid cell line has a transformed phenotype in vitro and al-tered and unlimited growth potential, conserving through itsability to differentiate. This cell line was initially used during the1990s by many teams around the world in signal-transductionstudies; however, later, the phenotype and the physiology of thesecells were shown to be different to human primary keratinocytesand therefore were considered not suitable for studies in skinphysiology (22–27). Moreover, the data of Numaga-Tomita andPutney (18) on calcium switch-induced differentiation involve theexpression of only one marker of differentiation, KRT1, which

cannot reflect differentiation per se, especially bearing in mindthat the expression of this protein is Ca2+-dependent (28).Because in our experiments Orai1 expression was found to

decrease in differentiated cells following calcium switch, wehypothesized that down-regulation of ISOC, which accompaniesthe transition to the differentiated state, is associated with thereduction of Orai1 levels. And indeed, our results allow us toconclude that the transition to the differentiated hPK phenotypeis associated with the loss/decrease of Orai1 expression and isunder its negative control.The principal question concerns the differential role of two

calcium entries in keratinocytes: SOCE and massive constitutiveentry. A number of calcium-permeable channels have beenshown to be expressed in hPK (29) especially those belonging tothe Transient Receptor Potential (TRP) family. From this TRPCfamily, TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 wereshown to be expressed and functional in hPK, and all of them wereshown to be involved in SOCE (27, 29–31). Interestingly, althoughthe short-term transient increase in [Ca2+]i is mostly attributed tobe proproliferative, for two of them, TRPC1 and TRPC4, whichwhere shown to be SOCs, a role in SOCE and keratinocyte dif-ferentiation was shown (27, 29, 32, 33). Among all of them,a member of the vanilloid family of TRP channels, TRPV6, hasbeen shown to be expressed and to positively correlate with thecalcium gradient (a stepwise increase in both intra- and extracel-lular calcium from stratum basale to stratum corneum) (34–36)

Fig. 6. Calcium influx via Orai1 activates FAK and triggersmigration. (A) A representative experiment of three in-dependent experiments of a wound-healing assay of UhPK(kept in 0.07 mM Ca2+) cells treated with 100 ng/mL EGF (+EGF)for the indicated time or without EGF (−EGF), and transfectedwith either siOrai1 or siCtrl. (B) Cell-migration assay usingTranswell chambers of UhPK (kept in 0.07 mM Ca2+) treatedwith 100 ng/mL EGF (+EGF) or without EGF (−EGF), and trans-fected with either siOrai1 or siCtrl. Cells were allowed to mi-grate for 24 h. Data shown are mean ± SEM; **P < 0.01, ***P <0.001; n = 3 in quadruplicate. (C) Cell-migration assay usingTranswell chambers of UhPK (kept in 0.07 mM Ca2+) treatedwith DMSO (CT), 10 nM iCalpI, 1 μM iCalpII, 1 μM BTP2, 10 μMML9, and 100 nM iPKCβ. Data shown are mean ± SEM; *P <0.05. (D) FAK397 autophosphorylation studies using UhPK cellstreated with 100 ng/mL EGF (+EGF) for 30 min or without EGF(−EGF), and pretreated with 1 μM BTP2, 1 μM Gö6976, 1 μMU73122 for 45 min. Numbers below correspond to the relativequantity of FAK397 compared with the total FAK, and theloading control β-actin. (E) The effects of pharmacological in-hibition of Orai1 (BTP2, 1 μM), STIM1 (ML9, 10 μM), PLCγ(U73122, 1 μM), and two PKC inhibitors (iPKCβ, 0.1 μM andGö6976, 1 μM) on FAK cleavage under the EGF stimulation(+EGF) yielding the expected cleaved form of FAK at around80 kDa. Numbers below correspond to the relative quantity ofthe cleaved FAK (80 kDa) compared with the FAK full-length(125 kDa), and the loading control β-actin. (F) Store-operatedcalcium entry in UhPK cells induced by 100 ng/mL EGF and itsinhibition by siOrai1. A representative experiment of five in-dependent experiments, n = 30 cells per experiment. (G) Store-operated calcium entry in UhPK cells induced by 100 ng/mL EGFand its inhibition by PLC inhibitor U73122, 1 μM.

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and differentiation in the skin, providing a massive calcium entryinto keratinocytes that is necessary to induce differentiation (37,38). The Orai1 channel and Orai1-mediated SOCE are likely tocontrol the onset of differentiation. Indeed, the existence of lipidrafts/caveolae as microdomains and initiation of calcium signalingtherefrom are well-known (39, 40). The formation of STIM1–Orai1complexes in those rafts was already demonstrated for SOCE (41).Although the disruption of lipid rafts resulted in an increase of theproliferative capacity of human keratinocytes, their reintegrationled to the expression of the late differentiation marker, KRT2 (42).It remains intriguing just how these two types of entries may coexistand whether they are interrelated because the decrease in SOCEand Orai1 expression is accompanied by the increase in constitutivecalcium entry and TRPV6 expression following the gradient ofcalcium and differentiation (37).We have demonstrated the important role of the Orai1 channel

in UhPK proliferation. A role of the Orai1 channel and SOCE incell proliferation has been suggested for endothelial cells (13),lymphocytes (14), airway smooth muscle (43), and others. Theincrease in Orai1 and STIM1 expression, and therefore SOCE ingeneral is a prerequisite to stimulate proliferation because the useof SOC inhibitors like SKF-96365, NiCl2, or BTP2 abrogated theobserved effects, as well as the selective knockdown of Orai1 andSTIM1 (43). The increase in [Ca2+]i induced by SOCE was shownto activate the proliferation-associated calcineurin-transcriptionfactor NFAT pathway (14, 44). Further studies will be needed tocorrelate the molecular mechanisms of Orai1-promoted pro-liferation and the calcium gradient in the skin.

In normal epidermis, keratinocytes migrate upward from thebasal layer as they undergo terminal differentiation, yet they alsohave the capacity for lateral movement during wound healing. Itwas shown that keratinocytes in low-calcium medium were lat-erally more motile than keratinocytes in normal medium (45).During calcium-induced stratification, hPK cells moved upwardfrom the basal layer by gliding over their neighbors and formingcontacts with other suprabasal cells (3). Of central importance,mostly for keratinocytes, is the ability to balance proliferationand differentiation, control adhesion to an underlying substratum,and, when necessary, remodel this substratum and migrate in adirected fashion. These functions are particularly central to themitotically active epidermal cell hPK, which must continuallyundergo proliferation, detachment, and differentiation in thecourse of homeostasis, and upon injury, proliferate and migratetoward and repair the wound.We have demonstrated the involvement of Orai1 channels in

keratinocyte polarized motility and migration. Indeed, the co-ordinated and dynamic regulation of adhesions is central for cellmigration in both normal and pathological processes (46). Cellmigration is initiated by forming protrusions, which are stabilizedby integrin-mediated adhesions that establish structural andsignaling linkages between the extracellular matrix and the actincytoskeleton. A prominent component involved in this regulationis FAK (47). FAK promotes cell migration by its capacity toorchestrate signals between integrin and growth factor receptors(47). Downstream of integrin or growth factor stimulation, FAKis phosphorylated at Tyr-397, which is an important binding site

Fig. 7. Keratinocyte physiology in Orai1 KO mice. (A) Capaci-tative calcium entry induced by 1 μM TG in keratinocytes fromorai1+/+, orai1+/−, and orai1−/− mice. (B) Proliferation of kerati-nocytes from orai1+/− and orai1−/− mice treated with 100 ng/mLEGF (EGF+) or vehicle (EGF−), measured using either WTS assay(C) or direct cell counting (D). Cell migration assay usingTranswell chambers from orai1+/− and orai1−/− mice treatedwith EGF (EGF+) or vehicle (EGF−). (E) Skin epidermis fromorai1+/+ and orai1−/− mice stained with anti-Orai1, anti-KRT5,anti-KRT10, and anti-LOR antibodies.

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for Src family kinases (48). There are numerous reports that thecontrol of FAK phosphorylation and cell morphology is a keyfactor involved in the formation of focal contacts that regulatecell attachment and detachment required for cell migration (49).Previous studies have demonstrated a critical role for FAK asa regulator of adhesion dynamics (50, 51), as well as SOCE in thecontrol of NFAT translocation to regulate cell migration (19). Inthe present work, we have shown that the depletion of the ER bythe EGF-triggered pathway, followed by the activation of Orai1channels and consequently SOCE, may lead to autophosphor-ylation of FAK at Tyr-397, thus stimulating keratinocyte migra-tion. Nevertheless, the mechanisms by which FAK regulates theassembly–disassembly of focal adhesions remain to be elucidated.EGF is an important growth factor that elicits cellular sig-

naling through an EGF receptor and affects cell migration inmany cell types (52). When EGF binds to EGFR, the receptordimerizes and is autophosphorylated, which subsequently leadsto the downstream activation of mitogen-activated protein ki-nase (MAPK), phosphatidyl 3-kinase (PI3-kinase), and phos-pholipase C pathways (53, 54). It has also been shown thatEGFR signaling acts through the TRPV3 channel regulatingkeratinocyte cornification (55). In our studies, we used EGF asa natural regulator for the processes studied, and we have shownthat EGF signaling in keratinocytes involves SOCE mediated byOrai1 channels.Some reports demonstrated that phosphorylation of FAK was

increased in EGF-stimulated cell spreading and migration (56).Others have shown that EGFR-mediated signaling induced thedephosphorylation of FAK during cell motility (57). Thus, mostinvestigators have examined either FAK phosphorylation or de-phosphorylation stimulated by EGFR-mediated signaling duringcell migration and wound healing. It was reported that EGFR-induced signaling pathways are necessary for cell migration (58).Thus, EGF is involved antagonistically in both FAK phosphoryla-tion and dephosphorylation with different mechanisms in a cell (59),and there is still a limited understanding of the mechanism linkinga cell attachment–detachment process and EGF stimulation.On the other hand, evidence has emerged supporting the role

of the calpain family of intracellular calcium-dependent pro-teases in regulating cell migration (60, 61). Calpains have beenproposed to regulate migration, at least in part through theirability to modulate the dynamics of adhesions (62). Numerouscalpain targets have been identified, some of which are proteinsthat are present in focal adhesions, including talin, paxillin, andFAK (63). It has been proposed that calpain cleavage may playa role in terminating FAK signaling by attenuating its kinaseactivity (64). It seems likely that different signaling pathways maybe activated by the same ligand such as EGF because it has beenshown that, in undifferentiated keratinocytes, IFN-inducibleprotein-9 activates μ-calpain downstream of a PLC-β3-mediatedcalcium flux whereas EGF uses the same ERK to M-calpaincascade that it triggers to converge at the disassembly of adhe-sion plaques (65). In our studies, we have confirmed that theselective inhibition of calpain, most probably calpain I, arrestsUhPK migration, suggesting the crucial role of this pathway infocal adhesion turnover and cell motility mediated by Orai1activation upstream of FAK. Thus, it is likely that proteolysis ofthese substrates contributes to the regulation of adhesion dy-namics and cell migration.We can summarize our findings on the role of Orai1/STIM in

keratinocyte migration: the activation of EGFR induces thestimulation of PLCγ and consequently the depletion of calciumin the endoplasmic reticulum, which induces the redistribution ofthe calcium sensor STIM1 (Fig. 8). The latter, in turn, trans-activates the Orai1 channel and induces store-operated calciumentry. This store-operated calcium entry promotes both theautophosphorylation of FAK via activation of calcium-dependentPKCβ and the degradation of FAK by calcium-dependent μ-calpainprotease, enhancing focal adhesion turnover and thereby stimu-lating polarized cell motility of human keratinocytes.

Our data obtained in humans are strongly supported by ourorai1−/− mouse model studies. Although the absence of Orai1protein in keratinocytes affects the SOCE in these cells, it maybe compensated by other store-operated channels. In addition,the proliferation of keratinocytes and particularly polarized mi-gration, both basal and induced, are significantly decreased. Theconsequences of orai1−/− deletion lead to impaired epidermis/barrier formation as a result of aberrant keratinocyte structuralprotein expression, suppressed proliferation, and, eventually,migration—all of them crucial for normal skin homeostasis.

Materials and MethodsCell Culture and Human Skin Sections. Human Primary Keratinocytes (hPK)were purchased from Invitrogen. Cells were cultured at 37 °C in a humidifiedatmosphere containing 5% CO2 in a keratinocyte serum-free medium(K-SFM) supplemented with 5 μg/mL recombinant epidermal growth factor(EGF) and 50 μg/mL bovine pituitary extract (BPE), containing 0.07 mM Ca2+.The medium was changed three times a week, and cultures were split bytreating the cells with 0.25% (vol/vol) trypsin (in PBS) for 5 min at 37 °Cbefore confluency. Throughout the text, the terms UhPK and DhPK are used.They imply that hPKs kept in 0.07 mM Ca2+ are called undifferentiated (UhPK)because their phenotype corresponds to undifferentiated proliferating cellsfrom stratum basale, or hPK cells treated with 1.7 mM Ca2+ for at least 3 d,which provokes an irreversible differentiation process and keratinocytes haveto be considered as differentiated (DhPK).

Orai1−/− Animals and Gene Targeting. Gene targeting of the Orai1 gene wasperformed by homologous recombination in B6/3 embryonic stem (ES) cellsderived from C57BL/6 mice (TaconicArtemis GmbH). Chimeric mice withtargetedOrai1 alleleswere generated by blastocyst injection of heterozygousOrai1neo/+ ES cell clones, as previously described (17). All mice were main-tained in specific pathogen-free barrier facilities at the La Jolla Institute forAllergy and Immunology (LIAI) and were used in accordance with protocolsapproved by the Center for Animal Resources and LIAI. All mice were gen-otyped by PCR. PCR-ready genomic DNA was extracted from tail biopsiesusing the QuickExtractDNA Extraction Solution (Epicentre) and then ampli-fied with the primers WT Forward 5′-GGGTGTGGCGTATGCAAATAACCT-3′,WT Reverse 5′-ACTCGAGCCGGTCTCCC-3′, KO Reverse 5′-TCGTACCACCTT-CTTGGGACTTGA-3′ (IDT), and Taq Choice (Applied Biosystems).

Mouse Primary Keratinocyte Culture. Mouse primary keratinocytes were iso-lated by dissecting the skin from orai1−/− mice using a standard protocol. Theremoved skin was washed in PBS completed with Penicillin/Streptavidin 1%(Gibco) and then fragmented and incubated in a vol/vol solution of Colla-genase I (8 mg/mL) and Dispase (5 mg/mL) (Gibco) for 3 h at 37 °C. ThenTrypsin is added during 15 min, and the mixture is filtered using a 40-μm

Fig. 8. The role of Orai1 in keratinocyte polarized motility. The activationof EGFR induces the activation of PLCγ and consequently the depletion ofcalcium in the endoplasmic reticulum, which stimulates the calcium sensorSTIM1. The latter, in turn, transactivates the Orai1 channel and inducesstore-operated calcium entry. This store-operated calcium entry promotesboth the autophosphorylation of FAK397 via activation of calcium-dependentPKCβ and the degradation of Focal Adhesion Kinase by calcium-dependentμ-calpain protease, enhancing focal adhesion turnover and thereby bothstimulating polarized cell motility of human keratinocytes.

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filter, centrifuged at 1,000 × g for 5 min, and finally resuspended in a De-fined Epidermal keratinocyte Medium CnT-07, PCT (CellNTec) and incubatedat 37 °C and 5% CO2 for a week. Then, cell medium was replaced by CnT-57,PCT (CellNTec) and changed every 3 d.

siRNA Cell Transfection. All siRNAs were synthesized by Eurogentec FranceSASU and are indicated in Table S1. Cells were transfected with 60 nM siRNAusing 6 μl of Hyperfect transfection reagent (Qiagen) following the manu-facturer’s instructions. The efficiency of cell transfections with the siRNAs foreach particular target has been validated using real-time quantitative PCR.

Immunohistochemistry. Paraffinized human skin sections were obtainedfollowing breast-reduction surgery after informed patient consent and theapproval of the ethical committee of the St. Vincent Hospital of Lille. Par-affin-embedded skin sections were subjected to conventional deparaffini-zation followed by antigen retrieval using citrate buffer at 95 °C in a waterbath. After saturation in the solution containing 1% BSA and 0.05% TritonX-100 in PBS-gelatin, the skin sections were incubated with the specificrabbit polyclonal anti-ORAI1 antibody (ProScience; 1/200), mouse monoclonalanti-STIM1 (BD Biosciences; 1/200), mouse monoclonal anti-KRT 10 (ChemiconInternational; 1/500), anti-Involucrin (Sigma-Aldrich ; 1/500), anti-Filaggrin (Abcam;1/500), and anti-phospho-FAK (pTyr397) (Sigma-Aldrich; 1/200) overnight at4 °C. Goat polyclonal anti-rabbit and anti-mouse peroxidase-conjugatedsecondary antibodies (Chemicon International; 1/200) were used. After rev-elation with diaminobenzidine (Sigma-Aldrich), images were analyzed usinga Zeiss Axioskope microscope (Carl Zeiss) and Leica Image Manager software(Leica Geosystems AG Heinrich).

Immunocytochemistry. Cells grown on glass coverslips were washed once withPBS and fixed in 3.5% paraformaldehyde in PBS. PBS-glycine (30 mM) + BSA(50 μl/mL) was used as a saturation buffer, and permeabilization wasobtained using 0.1% Triton X-100. The cells were washed again in PBS andsubjected to F-actin staining by the Phalloidin-tetramethylrhodamine B iso-thiocyanate (Fluka-Sigma-Aldrich) for 30 min at room temperature (1/1000).Then, a conventional immunostaining procedure was used for the detectionof focal adhesion proteins: mouse monoclonal anti-paxillin (Abcam; 1/200)and rabbit polyclonal anti-phospho-FAK (pTyr397) (Abcam; 1/400). AlexaFluor 546 or 488 goat anti-rabbit or anti-mouse IgGs (Molecular Probes;¼,000) were used as secondary antibodies. Fluorescence was analyzed ona Carl Zeiss Laser Scanning Systems LSM 510 connected to a Zeiss Axiovert 200Mwith a ×63 1.4 numerical aperture oil immersion lens at room temperature.

Quantitative Real-Time PCR. The quantitative real-time PCR of Orai1, STIM1,FLG, and HPRT mRNA transcript was done using MESA GREEN qPCR Mas-terMix Plus for SYBR Assay (Eurogentec) on the Bio-Rad CFX96 Real-Time PCRDetection System. The sequences of primers are indicated in Table S2. TheHPRT (hypoxanthine-guanine phosphoribosyltransferase) gene was used asan endogenous control to normalize variations in the RNA extractions, thedegree of RNA degradation, and variability in RT efficiency. To quantify theresults, the comparative threshold cycle method ΔΔCt and CFX ManagerSoftware v2.0 were used.

Western Blotting. hPK cells were treated with an ice-cold lysis buffer con-taining 10 mM Tris·HCl, pH 7.4, 150 mM NaCl, 10 mM MgCl, 1 mM PMSF, 1%Nonidet P-40, and protease inhibitor mixture from Sigma. The lysates werecentrifuged at 15,000 × g and 4 °C for 20 min, mixed with a sample buffercontaining 125 mM Tris·HCl, pH 6.8, 4% SDS, 5% b-mercaptoethanol, 20%glycerol, and 0.01% bromophenol blue, and boiled for 5 min at 95 °C. Thetotal protein samples were subjected to 8–10% SDS/PAGE and transferred toa PVDF membrane by semidry Western blotting (Bio-Rad Laboratories). Themembrane was blocked in a 5% milk TNT buffer (Tris·HCl, pH 7.4, 140 mMNaCl, and 0.05% Tween 20) for 1 h, and then probed using a specific rabbitpolyclonal anti-FAK (Abcam; 1/400) and anti-phospho-FAK (pTyr397) (Sigma-Aldrich; 1/1000) and anti-β-actin (Lab Vision; 1/1000) antibodies. The bandson the membrane were visualized by enhanced chemiluminescence (PierceBiotechnologies). A densitometric analysis was performed using a Bio-Radimage acquisition system (Bio-Rad Laboratories).

Electrophysiology and Solutions. Macroscopic currents were recorded fromhPK cells in the whole-cell configuration of the patch-clamp technique using

a computer-controlled EPC-9 amplifier (HEKA Electronic), as previously de-scribed. Voltage ramps (−100 to +100 mV) of 250 ms were recorded every 2 sfor IP3 and 5 s for TG immediately after gaining access to the cell froma holding of 0 mV. The currents were normalized based on the cell capaci-tance. Leak currents were subtracted by subtracting an initial ramp current(before SOC development) from all subsequent ramp currents.

The composition of the extracellular solution for patch-clamp recordingwas (in mM): 120 NaCl, 10 CaCl2, 2 MgCl2, 5 glucose, and 10 Hepes, pH 7.4(adjusted with TEA-OH), osmolarity 310 mOsm/kg adjusted with D-mannitol.The patch pipettes were filled with the basic intracellular pipette solution(in mM): 120 Cs-methane sulfonate, 10 CsCl, 10 Hepes, 10 BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid], and 6 MgCl2 (pH adjustedto 7.4 with CsOH and osmolarity 295 mOsm/kg adjusted with D-mannitol).The necessary supplements in the desired concentrations were added to theexperimental solutions directly from the appropriate stock solutions, dis-solved in water, ethanol, or dimethyl sulfoxide. All chemicals were pur-chased from Sigma-Aldrich. The experiments were carried out at roomtemperature.

Cell-Proliferation Assays. hPK cells were harvested and plated at the initialdensity of 3,000 cells per well in 96-well plates (Poly-Labo). Cells were trans-fected, and the number of viable cells was determined every 24 h using theCellTiter 96 Aqueous nonradioactive cell proliferation assay (Promega Corp.).Cell count was also used to determine the proliferation rate of hPK 48 h aftertransfection with siRNA using a Malassez counting chamber. Cells were tryp-sinized from six-well plates, and cells from each well were counted eight times.

Migration Assay. Cells were seeded onto the top of Transwell cell-cultureinserts with 8.0-μm pore size (Falcon) at the density of 20,000 per well (24-well format) in noncomplemented K-SFM. Cells were stimulated to migrateacross the filters by complemented K-SFM as a chemoattractant in the assaychambers beneath the inserts. After 24 h of incubation at 37 °C, non-migratory cells were removed from the top of the filter by scraping whereascells that had migrated through the filter pores to the lower side of theinserts then were fixed in methanol and stained with Hoechst (5 mg/L inPBS). Cells on the lower side of the inserts were counted using a LeicaDMIRB. Data are expressed as means of four wells ± SEM.

Wound-Healing Assay. hPK cells were seeded and allowed to reach subcon-fluency, and then cells were transfected with the siRNAs. After 24 h, a 1,000-μL pipette tip was used to scrape across the dish, and the resulting woundwas washed with PBS. Bright field images were captured along the woundusing a Nikon TS100 microscope (×100), and then a series of pictures weretaken at 24 h, 48 h, and 72 h after wounding. The images were then ana-lyzed using Adobe Photoshop CS5.

Cell Motility. hPKs were transfected with the corresponding siRNA for 24 h ortreated with the FAKi 14 (Tocris Bioscience; 10 μM) for 30 min. Cell membranewas stained for 30 min with CellMask, and cell movements were observed bytaking images every 36 s using a Carl Zeiss Laser Scanning Systems LSM 510connected to a Zeiss Axiovert 200 M.

Data Analysis. For each type of experiment, the data were accumulated fromat least three measurements. Electrophysiological data were analyzed offlineusing HEKA (HEKA Electronic) and Origin 7.0 (Microcal Software) software.Results were expressed as mean ± SEM, where appropriate. N equals thenumber in the series of experiments, and n equals the number of cells usedin the study. ANOVA was used for statistical comparison of the differences,and P < 0.05 was considered significant. In the graphs, (*) and (**) denotestatistically significant differences with P < 0.05 and P < 0.01, respectively.

ACKNOWLEDGMENTS. We thank Dr. Adriana Mihalache and Prof. PierreGosset for immunostaining expertise. We also thank Dr. David House fromGlaxoSmithKline for the provision of GSK-7975A and GSK-5503A compounds.We thank the Department of Research and Development of Johnson andJohnson France for the PhD funding of M.V. and M.R., as well as for thescientific collaboration with our laboratory. D.G. was supported by the StateFund for Fundamental Research (F 46.2/001). The studies on keratinocytesfrom Orai1−/− mice were funded in part by National Institutes of HealthGrants AI084167 and AI40127 (to A.R. and P.G.H.).

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E4848 | www.pnas.org/cgi/doi/10.1073/pnas.1310394110 Vandenberghe et al.

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