Experimental Cell Research 266, 250–259 (2001)doi:10.1006/excr.2001.5215, available online at http://www.idealibrary.com on

Expression of the Helix–Loop–Helix Protein ID1 in Keratinocytes IsUpregulated by Loss of Cell–Matrix Contact

Birgit M. Schaefer,*,1 Judith Koch,* Alexander Wirzbach,† and Michael D. Kramer†

*Institute for Immunology, Im Neuenheimer Feld 305, D-69120 Heidelberg, Germany; and †Lynx Therapeutics GmbH,

Im Neuenheimer Feld 515, D-69120 Heidelberg, Germany

To analyze the inhibitor of DNA-binding type 1 (ID1)in the human epidermis and in cultured keratinocyteswe generated and characterized ID1-specific monoclo-nal antibodies. Immunohistological studies on humanskin biopsies revealed that ID1 is not detectable innormal human epidermis but in lesional epidermis ofbullous pemphigoid. In the latter case we found ID1 inthe cytoplasm of basal and proximal suprabasal kera-tinocytes. Cultured normal human epidermal keratin-ocytes displayed ID1 in the cytoplasm; upon differen-tiation into a multilayered keratinocyte sheet, ID1 wasno longer detectable. It was reexpressed after dispase-mediated detachment of the keratinocyte culturesfrom the growth substratum. In this case ID1 was lo-calized to the cytoplasm and the nucleus. Our dataindicate that after epidermal injury—in our case lossof cell–matrix contact—ID1 is upregulated in affectedkeratinocytes. In view of the ID1 function in other celltypes, we speculate that ID1 facilitates the transitionfrom the resting to the migrating and proliferatingkeratinocyte required for efficient repair of epidermallesions by reepithelialization. Taken together we sug-gest that ID1 is an important player in epidermal(patho-)physiology. © 2001 Academic Press

INTRODUCTION

Helix–loop–helix (HLH) proteins are a family ofdimeric transcription factors involved in the regulationof cell growth and differentiation [1, 2]. Four classes ofHLH proteins have been described, classes A–D, whichall share the HLH motif—a conserved domain respon-sible for DNA binding and homo/heterodimerization [3,4]. In classes A–C, an additional basic domain at theN-terminus of the HLH motif is found; these HLHproteins are therefore termed basic HLH proteins(bHLHs). The basic domain together with parts of theloop and the second helix mediates DNA binding. Bind-ing interactions occur between various bHLH members

1 To whom reprint requests should be addressed. Fax: 1149/6221/

565595. E-mail: bo4@ix.urz.uni-heidelberg.de.

2500014-4827/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

of classes A–C to form hetero- and/or homodimers[3–5]. It is only in this dimeric form that bHLH-tran-scription factors bind to DNA [e.g., 6].

Class A bHLH proteins are ubiquitously expressed,while class B bHLH proteins are expressed in a tissue-specific manner. Classes A and B bHLH proteins arenegatively regulated by members of the class D HLHproteins the so-called inhibitors of DNA binding, whichare synomously termed IDs. Four IDs are known, IDs1–4. IDs 1–3 are ubiquitously expressed, while ID 4 isexpressed in a tissue-specific manner. In contrast toclasses A–C HLHs, IDs lack the N-terminal basic re-gion. Heterodimerization of class A or B bHLH proteinswith IDs results in a dimer that cannot bind to DNAand is therefore functionally inactive [7, 8].

Data on the role of HLH proteins in the (patho-)-physiology of epidermal keratinocytes are rare: Mad, abHLH class C protein, has been found in the supra-basal keratinocytes of the murine epidermis and issuggested to participate in the control of epidermaldifferentiation and proliferation [9]. Furthermore, thekeratinocyte dioxin receptor belongs to the bHLH fam-ily and it binds to DNA only after interaction with itsdimerization partner Arnt [10]. Dermo-1 a twist-re-lated class B bHLH protein is expressed in the devel-oping murine dermis [11]. Overexpression of IDs 1–3extends the life span of primary human keratinocytesin vitro and reduces their differentiation capacity [12],which supports the notion that IDs function as inhibi-tors of keratinocyte differentiation and promoters ofkeratinocyte growth. No data are available on thephysiological expression of IDs in epidermal keratino-cytes in vivo.

In view of the finding that ID1 has the most pro-nounced effect on keratinocyte differentiation [12], wefocused on the analysis of ID1 expression in normaland lesional human epidermis. We generated and char-acterized monoclonal anti-ID1 antibodies to be used forimmunohistology. Using these antibodies we investi-gated ID1 expression in normal and lesional epidermis(bullous pemphigoid) as well as in cultured keratino-cytes during differentiation [13]. Furthermore we stud-

ied ID1 in keratinocyte cultures that were enzymati-

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251ID1 EXPRESSION IN EPIDERMAL KERATINOCYTES

cally detached from their growth substratum, anexperimental approach that at first approximationmimics epidermal lesion formation in subepidermalblistering diseases like in bullous pemphigoid [14].

MATERIAL AND METHODS

Cos cell culture and transfection. Cos cells were grown in RPMI-medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamin and 1% penicillin/streptomycin (Sigma, No. P-0701,Deisenhofen, FRG). Cells were passaged using an EDTA/trypsinsolution (Sigma, No. C-0376) for 10 min at 37°C. The day beforetransfection, 4 3 106 cells were seeded into petri dishes (14 cmdiameter). The next day cells were washed with phosphate-bufferedsaline (PBS) and incubated with the plasmid containing DEAE–dextran–chloroquine solution (20 mg plasmid, 144 ml DEAE–dext-ran, and 360 ml chloroquine in 18 ml RPMI/2% fetal calf serumFCS)) for 4 h at 37°C in a humidified atmosphere of 7% CO2.

Afterward the solution was aspirated and cells were incubated in 18ml RPMI/10% dimethylsulfoxide (DMSO) for 2 min. Cells werewashed in PBS two times and incubated in FCS- and L-glutamine-supplemented RPMI medium for 36–48 h at 37°C in a humidifiedatmosphere of 7% CO2. Cells were harvested using a rubber scraperand stored at 220°C until use. In case of immunocytology cos cellswere seeded onto glas slides. Transfection was performed as detailedabove. After the incubation period the glas slides were removed fromthe petri dishes fixed in acetone (10 min at room temperature) andstained immediately.

Keratinocyte culture and activation. Human keratinocytes wereobtained from skin biopsies via overnight trypsinization at 4°C. Cellswere cultured using the feeder-layer technique described by Rhein-wald and Green [13, 15] under differentiating conditions in petridishes (14 cm diameter) for 8 days in Dulbecco’s modified Eagle’smedium (DMEM) containing 10% fetal calf serum and supplements.Using this culture regimen keratinocytes become multilayered, aprocess that goes along with differentiation and growth arrest. Mul-tilayered cultures were treated for 30 min with dispase II (2.4 mg/mlin DMEM without FCS), washed twice in DMEM, and incubated fordifferent intervals of time (0, 4, 8 h) in complete DMEM. Dispasetreatment resulted in the detachment of the complete keratinocyteculture from its growth substratum. These detached keratinocytecultures were referred to as keratinocyte “sheets.” To avoid theinfluence of fresh serum, which is known to induce several genes[16], the keratinocyte cultures were fed 24 h prior to dispase treat-ment, and after dispase treatment keratinocyte sheets were incu-bated in the “old” medium directly collected before dispase treatment[14, 18]. To analyze nondifferentiated keratinocytes, keratinocyteswere seeded into six-well plates under the same culture conditions asmentioned above. Keratinocyte culture was stopped before cellsreached confluency (i.e., 3 days after seeding), thus avoiding forma-tion of a multilayered keratinocyte sheet.

HaCaT cells. HaCaT cells were cultured at 37°C in a humidifiedatmosphere of 7% CO2 using DMEM supplemented with 2 mML-glutamine and 10% (v/v) heat-inactivated fetal calf serum. Cells

ere grown to confluency. The medium was then switched to highalcium (1.5 mM) to induce differentiation. Cells were harvested 3ays later. The dispase treatment of HaCaT cells was performed asescribed for the primary human keratinocyte cultures. Cyclohexi-ide treatment of HaCaT sheets was performed after dispase de-

achment: sheets were incubated in 10 mg cycloheximide/ml mediumSigma, No. C 7698) for 8 h, harvested, and anlyzed immunohisto-ogically.

Cell extracts. Keratinocyte cultures prior to dispase treatmentere washed in PBS and scraped with a rubber policeman in PBS.eratinocyte cultures after dispase treatment (5keratinocyte

heets) were transferred into a 1.5-ml tube and washed with PBS. a

heets were stored at 220°C until use. Lysis was performed at 4°Csing HB buffer (0.3 M sucrose, 10 mM Tris, pH 8.0, 10 mM NaCl, 3M MgCl2, 0.5% NP-40, 13 protease inhibitor cocktail (Complete,

No. 1697498, Boehringer Mannheim GmbH, Mannheim, FRG)) for1 h. The lysate was then centrifuged at 13,000g for 15 min and thesupernatant was recovered and used for Western blotting and im-munprecipitation procedures.

Immunohistological/immunocytological staining. The detachedkeratinocyte resp. HaCaT sheets (either directly after detachment orafter 8 h of incubation) were snap frozen in liquid nitrogen andstored at 280°C until serial frozen sections of 4–5 mm were cut. Thesections were fixed in acetone, air dried, and stained with monoclo-nal antibodies HDpKeID1 5.21 and 210.12 as described previously[18]. Briefly, the sections were incubated with the primary antibody,which then was detected by using biotin-labeled goat anti-mouse IgGantibodies and Cy3-labeled streptavidin. Negative controls werePBS, or isotype-matched monoclonal mouse IgG instead of the firstantibody. For counterstaining of nuclei, sections were incubated withTOTO-3 (1:1000 in PBS) (No. T3604, Molecular Probes, Eugene, OR)for 10 min. Sections were mounted in PBS/glycerol and analyzedusing our confocal laser scanning system (Leica TCS NT laser scan-ning system, Leica, Bensheim, FRG).

In the case of immunocytology cos cells were grown on glass slides,transfected, and analyzed 2 days thereafter. Staining proceduresfollowed the above-mentioned staining protocol.

RNA preparation and Northern blot analysis. Total cellular RNA(0, 2, 4, and 8 h) was extracted from keratinocyte/HaCaT sheetsusing an RNA-extraction kit (No. CS-105, RNAZol B, WAK-ChemieMedical GmbH, Bad Soden, FRG) based on the acid guanidiniumthioglycolate–phenol–chloroform extraction method [19] and quan-tified by measuring absorbance at 260 nm. RNA (5 mg/lane) wassize-fractionated on 1.2% agarose/2.2 M formaldehyde gels, blottedonto nylon membranes (No. RPM 203N, Amersham Life Science,Little Chalfont, UK) by vacuum blotting, and cross-linked to thefilter by exposure to UV irradiation. Membranes were prehybridizedin Church buffer (0.25 M Na2HPO4, pH 7.2, 0.5 EDTA, 7% SDS) for

h. 39-Truncated ID1a (suitable for detecting ID1a and ID1b) sub-loned into the TA-TOPO vector was used for probe preparation.robes were labeled with [a-32P]dCTP by using the random primed

abeling kit (No. 1004760; Boehringer-Mannheim) and hybridized tohe immobilized RNA in Church buffer at 67°C overnight. The mem-ranes were then washed at high stringency (23 standard sodiumitrate (SSC)/1% SDS; 0.23 SSC/1% SDS) and exposed to KodakAR films at 280°C with an intensifier screen (Dr. Goos-Suprema,eidelberg, FRG).All filters were rehybridized with a GAPDH probe (No. 57090,TCC, Rockville, MD). The mRNA and GAPDH autoradiogramsere scanned with an image master scanning system (No. 56-1153-4, Amersham Pharmacia Biotech Europe GmbH; Freiburg, FRG).ensitometric measurement for the mRNA band was normalized to

he corresponding GAPDH band.cDNA synthesis and polymerase chain reaction (PCR). cDNA-

ynthesis and polymerase chain reaction were performed accordingo the manufacturer’s instructions using the Advantage RT-for-PCRit (No. K1402-2, Clontech, Palo Alto, CA) and the Advantage-GC 2CR Kit (No. K1913-1, Clontech), respectively.The following PCR primers were used for the amplification of

D1a/b:

1. ID1 59 (59-ATCATGAAAGTCGCCAGTGGCAG-39) and ID-39/159-AACACACGAGTGGAATCCCACCCCC-39), resulting in a 710-bpragment (5ID1a) and a 910-bp fragment (5ID1b);

2. ID1-59(59-ATCATGAAAGTCGCCAGTGGCAG-39) and ID1-39/259-GCGACACAAGATGCGATCGTCC-39), resulting in a 480-bpragment (5ID1a);

3. GST-ID1-59 (59-ATGGATCCATGAAAGTCGCCAGTGGCAG-39)

nd GST-ID1-39 (59-ATGAATTCTCAGCGACACAAGATGCGATCG-

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252 SCHAEFER ET AL.

39), resulting in a 480-bp fragment (5ID1a) with a 59-BamHI site anda 39-EcoRI site;

4. ID2-59 (59-AGCATGAAAGCCTTCAGTCC-39) and ID2-39 (59-TGAACACCGCTTATTCAGCC-39), resulting in a 430-bp fragment(5ID2);

5. ID3-59 (59-TCACTCCCCAGCATGAAGG-39) and ID3-39 (59-AG-GACACGGCCGAGTCAG-39), resulting in a 360-bp fragment(5ID3);

6. ID4-59 (59-GGGTTCGCTCGCGTAGAG-39) and ID4-39 (59-GCT-CAGCGGCACAGAATG-39), resulting in a 520-bp fragment (5ID4).

All PCR-products were cloned into the pcDNA3.1/V5/His-TOPOvector according to the manufacturer’s instructions (EukaryoticTOPO TA Cloning Kit, Invitrogen, Carlsbad, CA). In case of theGST-tagged ID1a fusion protein the PCR product was first clonedinto the pcDNA3.1/V5/His-TOPO vector. The fragment was thenreleased via BamHI/EcoRI digestion and ligated into the pGEX-2Tvector (No. 27-4801-01, Amersham Pharmacia Biotech Inc., Piscat-away, NJ).

ID1-, ID2-, and ID3-PCR-cDNAs were subcloned into the pIVEX2.4 vector for expression of the respective His-tagged recombinantprotein in the rapid translation system RTS-500 (No. 3018008, RocheDiagnostics GmbH–Roche Molecular Biochemicals, Mannheim,FRG).

Real-time quantitative rt-PCR. cDNA was synthesized from totalRNA of HaCaT-sheets directly after (T0) and 4 h after (T4) dispasetreatment using supercript II (No. 18089-011, Gibco BRL;) and anoligo(dT)24 primer (MWG-Biotech, Martinsried, FRG). The cDNAswere then measured via real-time quantitative PCR using the ABIPrism Model 7700 (Applied Biosystems, Weiterstadt, FRG) and theSYBR-Green I dye master mix (No. 4309155, Applied Biosystems)according to the manufacturer’s protocol. In brief, SYBR-Green in-tercalates into double-stranded DNA, i.e., the PCR product. By mea-suring the increased fluorescence direct detection of the PCR productis acchieved. Forward and reverse primers (each at a concentrationof 300nM) were ID1-for (59-ACGTGCTGCTCTACGACATGA-39) andID1-rev (59-GGATTCCGAGTTCAGCTCCAA-39). For quantitationthe comparative CT method was choosen. With this method, theamount of target (T4), normalized to an endogenous reference (b-actin) and relative to a calibrator (T0) can be deduced. For detailsrefer to User Bulletin 2 from Applied Biosystems. To ensure specifityof the PCR reaction, gel electrophoresis was performed.

Expression and affinity purification of recombinant GST-ID1a.Expression and affinity purification of GST-ID1a fusion protein wasperformed according to the manufacturer‘s instructions. Briefly,pGEX-2T-ID1a-positive Escherichia coli was grown in ampicillincontaining LB medium (50 mg/ml) overnight. The overnight culture

as diluted and grown to log phase (OD 0.6). Gene expression wasnduced with isopropyl b-D-thiogalactoside (IPTG) (0.1 mM end con-entration). Cells were grown for another 2 h at 37°C and thenarvested by centrifugation. The cell pellet was resuspended in PBSnd cells were lysed by sonification. Cell debris was removed fromhe supernatant by centrifugation. The sonicated fusion protein su-ernatant was affinity purified using a glutathione–Sepharose 4Bolumn (No. 17-0756-01, Amersham Pharmacia Biotech Inc., Pisca-away, NJ). The GST-ID1a fusion protein was eluted using glutathi-ne elution buffer (10 mM reduced glutathione in 50 mM Tris–HCl,H 8.0) and analyzed via SDS–PAGE and Western blotting.The recombinant GST-ID1a fusion protein was used for immuni-

ation of rabbits (Dr. J. Pineda Antikorper-Service, Berlin, FRG) andice, as detailed elsewhere [e.g., 20].Expression of ID1, ID2, and ID3 in the RTS system. ID1, ID2, and

D3 recombinat proteins were expressed in the rapid translationystem RTS-500 (No. 3018008, Roche Diagnostics GmbH–Roche Mo-ecular Biochemicals) according to the manufacturers’ instructions.n brief, 1 ml reaction solution (0.25 ml E. coli lysate, 0.75 ml

eaction mix, 50 ml energy mix, up to 50 ml DNA template (10 mg

lasmid–DNA)), and 10 ml feeding solution (10.5 ml reconstitutedeeding mix, 0.5 ml energy mix) were loaded in the reaction respec-ive feeding compartment of the reaction device. Reaction was thenerformed overnight at 30°C in the RTS-500 instrument. Reactionolution was stored at 220°C until use.Electrophoresis and Western blotting. Polyacrylamide gel electro-

horesis was performed according to the procedure of Laemmli with13% resolving gel and a 3% stacking gel. Resolved proteins in cell

xtracts were transferred electrophoretically to nitrocellulose mem-ranes (Hybond-C extra, No. RPN303E Amersham Life Science) forh at 250 mA. The nitrocellulose was blocked in Tris-buffered saline

TBS)/5% milk powder for 2 h at room temperature. The membraneswere then incubated with the first antibody (undiluted supernatantHDpKeID1 5.21, HDpKeID1 10.12, mouse anti-His 1:500 in TBS/3%milk powder (No. R930-25, Invitrogen, Groningen, Netherlands),rabbit anti ID1 1:10,000 in TBS/3% milk powder, goat anti GST1:5000 in TBS/3% milk powder (No. 27-4577-01, Amersham Phar-macia Biotech Inc., Piscataway, NJ)) overnight at 4°C. The nitrocel-lulose was washed two times in TBS/0.2%Tween 20, one time in TBS,and then incubated in the second antibody (1:10,000 TBS/3% milkpowder) peroxidase-conjugated anti-mouse IgG, anti-rabbit IgG,anti-goat IgG (No. 115-035-003, No. 111-036-046, and No. 305-036-003, Dianova, Hamburg, FRG). Nitrocellulose was washed onceagain and visualization was performed using a chemiluminescencedetection system (ECL Western blotting detection reagents, No. RPN2106, Amersham Pharmacia Biotech Inc., Piscataway, NJ).

Immunoprecipitation. Using the HB buffer cell extracts contain-ing recombinant/native ID1 were immunoprecipitated with our poly-clonal anti-ID1a serum. Cell extracts were precleared with proteinG–Sepharose (protein G–Sepharose 4 Fast Flow, No. 17-0618-01,Amersham Pharmacia Biotech AB, Uppsala, Sweden). The cell ex-tract was incubated overnight at 4°C with the rabbit antiserum.Protein G–Sepharose was then added followed by 2 h incubation at4°C. The Sepharose beads were collected by centrifugation and thepellet was washed three times in HB buffer. To dissociate ID1a fromthe Sepharose beads the pellet was resuspended in 100 ml SDS–lysisuffer and boiled for 5 min. The released materials were analyzed byDS–PAGE and Western blotting.Nonradioactive cycle sequencing. Plasmid DNA was sequenced

sing the ABI-310 sequencer from Perkin Elmer. Sequence reactionsere performed according to the manufacturer‘s instructions using

he DNA sequencing kit (No. 4303153, Perkin Elmer Applied Bio-ystems, Warrington, UK).

RESULTS

Production and Characterization of Monoclonal antiID1a Antibodies

We tested cDNA of the keratinocyte cell line HaCaTfor expression of ID1 via PCR using the primer pairID1-59 and ID1-39/1. HaCaT cells were found to expressoth ID1 splice variants, i.e., ID1a and ID1b [21, 22]data not shown).

For the expression of recombinant ID1 the followingonstructs were generated by PCR using the primerairs ID1-59 and ID1-39/1, ID1-59, and ID1-39/2 as wells GST-ID1-59 and GST-ID1-39: (A) ID1a and ID1b

subcloned into the TA-TOPO vector allowing the eu-caryotic production of untagged recombinant ID1a andID1b, (B) ID1a subcloned into the TA-TOPO vector,allowing eucaryotic production of V5/His-tagged re-

combinant protein, and (C) ID1a subcloned into the

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253ID1 EXPRESSION IN EPIDERMAL KERATINOCYTES

pGEX-2T vector for bacterial expression of GST-taggedrecombinant protein. All constructs were verified bynonradioactive cycle sequencing [23].

The GST-tagged ID1a fusion protein was prepared,affinity purified, and used for immunization of miceand rabbits as detailed in material and methods.Antibodies purified from rabbits and hybridomas re-acting specifically with the GST-ID1a fusion proteinwere selected for further characterization: Westernblotting was performed on cos-cell-lysates trans-fected with V5/His-tagged and untagged ID1a. Un-treated cos-cell-lysate was used as negative control.The rabbit antiserum as well as our panel of mono-clonal antibodies (e.g., HD pKeID1 5.21 and 210.12)stained a band of approximately 21 kDa in the ly-sates obtained from the V5/His-tagged ID1a-trans-fected cos cells. Only weak reactivity with a band ofapproximately 16 kDa was obtained using the lysateof cos cells transfected with recombinant ID1a. Noreactivity was observed on untransfected cos-cell ly-sates. Since it is suspected that the amount of recom-binant IDs in cos cells is rather low due to their shorthalf-lives [24, 25], and to confirm reactivity of ourantibodies with recombinant ID1a, Cos cells weretransfected with V5/His-tagged or untagged recom-binant ID1a and immunoprecipitated using the rab-bit antiserum. As depicted in Fig. 1A the monoclonalantibodies HDpKeID1a 5.21 and 210.12 stained a21-kDa protein as did our positive control antibodyagainst the V5 tag. In case of the untagged recombi-nant ID1a a protein of 16 kDa was detected by bothantibodies (Fig. 1B). No reactivity was found on con-trol immunoprecipitates using rabbit IgG instead ofanti-pKeID1a rabbit IgG.

ID1a and ID1b are largely homologous splice vari-ants implicating that our anti-ID1a monoclonal anti-bodies cross-react with ID1b. To elucidate cross-reac-tivity, cos cells were transfected with recombinantID1b and subjected to SDS–PAGE and Western blot-ting. The polyclonal antiserum as well as our monoclo-nal antibodies HDpKeID1a 5.21 and 210.12 (Fig. 1C)cross-reacted with ID1b.

To exclude reactivity of our monoclonal antibodieswith other members of the ID-family rtPCR usingcDNA from HaCaT cells and keratinocyte cultures be-fore and 8 h after dispase treatment was performed toanalyze ID2-, ID3-, and ID4-mRNA expression. HaCaTcells and keratinocytes before dispase treatment werefound to express ID2- and ID3-specific mRNA, afterdispase treatment no ID3-specific PCR product wasobserved. ID4-specific mRNA was detected in neitherHaCaT cells nor keratinocytes (data not shown). ID1-,ID2-, and ID3-specific cDNAs were then (sub-)clonedinto the pIVEX 2.4 vector. Recombinant N-terminalHis-tagged ID1-, ID2-, and ID3-protein were generated

with the help of the RTS in vitro translation system.

TS lysates were subjected to SDS–PAGE and West-rn blotting procedures using our monoclonal antibod-es HDpKeID1a 5.21 and 210.12 as well as a monoclo-al antibody against the His tag to ascertainxpression of ID2 and ID3. All three recombinant pro-eins were found to be expressed in the RTS system;owever, our monoclonal antibodies HDpKeID1a 5.21nd 210.12 only reacted with the His-tagged ID1 butid not crossreact with recombinant ID2 or ID3 (dataot shown).To test the reactivity of the antibodies HDpKeID1a

.21 and 210.12 in immunocytology, cos cells weretransfected with untagged recombinant ID1a. Both an-tibodies displayed reactivity with the transfected coscells (Fig. 2), whereas no reactivity was observed innontransfected cos cells.

Taken together, our data indicate that the monoclo-nal antibodies HDpKeID1a 5.21 and HDpKeID1a10.12 both react equally well with the two human ID1splice variants ID1a and ID1b when used in Western

FIG. 1. (A) Cos cells were transfected with V5/His-tagged ID1a.Immunoprecipitation was performed as detailed under Materialsand Methods. The precipitate was subjected to SDS–PAGE andWestern blotting was performed using the mabs anti-V5 (lane 1),HDpKeID1a 5.21 (lane 2), and HDpKeID1a 10.12 (lane 3). MouseIgG (lane 4) was used as negative control. Anti-V5 and HDpKeID1a5.21 and 210.12 reacted with the 21-kDa V5/His-tagged ID1a pro-tein. (B) Cos cells were transfected with untagged ID1a. Immuno-precipitation and Western blot was performed. The antibodiesHDpKeID1a 5.21 (lane 1) and 210.12 (lane 2) reacted with the16-kDa recombinant ID1a. Mouse IgG (lane 3) was used as negativecontrol. (C) To elucidate the reactivity of our monoclonal antibodieswith ID1b, cos cells were transfected with untagged recombinantID1b. Both antibodies displayed reactivity with recombinant ID1b(15.5 kDa). The reactivity of HDpKeID1a 5.21 is depicted in lane 1;the reactivity of HDpKeID1a 10.12 in lane 2; control mouse IgG inlane 3.

blot and immunocytology techniques.

254 SCHAEFER ET AL.

ID1-Expression in Normal Epidermis and LesionalSkin of Bullous Pemphigoid

To analyze ID1 expression in normal epidermis anddiseased epidermis of five patients with bullous pem-phigoid, immunohistology was performed using themonoclonal antibody HDpKeID1a 5.2. In normal epi-dermis, ID1-expression was not detectable (Fig. 3A).However, in lesional epidermis of bullous pemphigoid(three of five cases) we obtained evidence for ID1 ex-pression: throughout the lesional epidermis basal andsuprabasal keratinocytes showed a punctated cytoplas-matic staining. ID1-expression in these keratinocyteswas restricted to the cytoplasm and was not observedin the nucleus (Fig. 3B). The cytoplasm of keratino-cytes of the upper stratum spinosum and the stratumcorneum of lesional epidermis of bullous pemphigoidwas negative for ID1. In addition we investigated dis-eased human skin suspected to have discoid or sys-temic lupus erythematodes (three patients), collageno-ses (two patients), dermatitis herpetiformis Duhring(two patients), leucocytoclastic vasculitis (two pa-tients), and psoriasis (three patients). In all lattercases no ID1-staining was observed in lesional epider-mis. Taken together, these data indicate that our mabHDpKeID1a 5.21 detects native ID1 in immunohistol-ogy and that ID1-protein is upregulated in keratino-cytes involved in subepidermal blister formation.

ID1-Expression in Cultured Normal EpidermalKeratinocytes (NHEK) and in the Keratinocyte CellLine HaCaT

To test the hypothesis that ID1 is upregulated uponloss of cell matrix contact, we performed immunohis-tological studies on NHEK and HaCaT sheets that hadbeen enzymatically detached from their growth sub-stratum, an experimental approach that at first ap-proximation mimics epidermal–dermal dyshesion andthat we have extensively characterized recently [14,18]. We compared NHEK and HaCaT sheets directlyafter detachment and 8 h thereafter. No positive stain-

FIG. 2. Cos cells were grown on glass slides, transfected withrecombinant untagged ID1a, and stained with the mabs HDpKeID1a5.21 (A) and 210.12 (B) as detailed under Materials and Methods.Nuclei were counterstained with TOTO-3. Stainings were analyzedusing a laser scan microscope. Positive ID1a-staining reactions aredepicted in green, the nuclei appear in red.

FIG. 3. Cryostat sections of normal skin (A) and a biopsy derivedfrom diseased skin of a bullous pemphigoid patient (B) were analyzedfor the expression of ID1 using the mab HDpKeID1a 5.21 (see Ma-terials and Methods). Stainings were analyzed using a confocal laserscanning microscope. Normal skin/epidermis was found to be ID1-negative. In case of the biopsy from the bullous pemphigoid patientbasal and suprabasal keratinocytes were found to express ID1, de-picted in green, in the cytoplasm. Nuclei were counterstained with

TOTO-3. e, epidermis; l, lumen of the blister; d, dermis.

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255ID1 EXPRESSION IN EPIDERMAL KERATINOCYTES

ing could be demonstrated directly after dispase treat-ment (Fig. 4A), whereas a weak cytoplasmtic and astrong nuclear staining was observed in NHEK (Fig.4B) and HaCaT sheets 8 h after dispase treatment.

For further analysis lysates prepared from NHEKnd HaCaT sheets before and 8 h after detachmentere subjected to SDS–PAGE and Western blotting.rior to detachment ID1 was undetectable in NHEK

ysates, whereas 8 h after dispase treatment ID1 ('16

FIG. 4. Normal human epidermal keratinocytes (NHEK) were gdetached from the growth substratum using dispase as detailed undetreatment (A) and 8 h thereafter (B). Cryostat sections were analyzedin green, was found to be expressed in keratinocyte sheets 8 h afterstrong signal was found in the nuclei of keratinocytes (arrows in B).be ID1-negative. Nuclei were counterstained with TOTO-3 and are

FIG. 6. Keratinocytes were grown to semiconfluency and stainedmicroscopy. Nearly all keratinocytes were found to express ID1 (dTOTO-3 and are depicted in red.

Da protein) was clearly detectable (Fig. 5A). In Ha- a

aT cells ID1 could not be demonstrated using simpleestern blotting technique (Fig. 5A). However, immu-

oprecipitation of native ID1 in HaCaT cells prior tond 8 h after dispase treatment revealed upregulationf ID1 in HaCaT cells upon detachment (Fig. 5B). Ourndings on differentiated primary human keratino-ytes as well as on the keratiocyte cell line HaCaTrovide evidence that (1) both of our monoclonal anti-odies detect native ID1 in Western blotting technique

n to confluency under differentiating conditions. The sheets wereaterials and Methods. Sheets were harvested directly after dispase

the expression of ID1 using the mab HDpKeID1a 5.21. ID1, depictedachment (B). The protein was weakly expressed in the cytoplasm. Aratinocyte sheets directly after dispase treatment (A) were found toicted in red.h mab HDpKeID1a 5.21. Stainings were analyzed by laser scanningcted in green) in the cytoplasm. Nuclei were counterstained with

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256 SCHAEFER ET AL.

tachment of keratinocytes from their growth substra-tum.

To explore whether ID1 is downregulated upon dif-ferentiation in vitro we compared immunohistologicaltainings of cultured normal human keratinocytesith our stainings of multilayered keratinocyte sheetsirectly after dispase treatment. In contrast to theultilayered keratinocyte sheet, keratinocytes grown

s monolayers express ID1. ID1 expression was inde-endent of the confluency status. The staining ap-eared to be located in the cytoplasm, the nuclei wereD1-negative (Fig. 6).

Taken together, data indicate that keratinocytes ex-ress ID1 in vitro, downregulate ID1 upon differentia-ion, and upregulate ID1 again upon detachment fromheir growth substratum. Furthermore, detachment oferatinocytes appears to involve translocation of ID1nto the nucleus.

etection of ID1a/b-Specific mRNA in NHEK andHaCaT Cells prior and after Dispase Detachment

To explore whether the ID1 upregulation uponispase treatment is due to an increased expressionf specific mRNA we performed Northern blottingxperiments. RNA from untreated and dipase-de-ached NHEK and HaCaT cells (directly after as wells 4 and 8 h after dispase treatment) was isolatednd subjected to Northern blotting. Hybridizationith a ID1-specific probe revealed two bands, 1100nd 880 bp in size (data not shown), according to thenown ID1a- and ID1b-specific mRNA sizes, respec-ively. Blots were rehybridized with GAPDH (dataot shown). Densitometric analysis of the ID1a/b andAPDH bands revealed no evidence for mRNA reg-lation of ID1a or ID1b upon dispase detachment ofHEK/HaCaT cells, indicating a (post-)translational

egulation mechanism for ID1 expression in keratin-cytes under these conditions. These data were fur-her corroborated by quantitative rtPCR on cDNArom HaCaT sheets directly after and 4 h after dis-ase treatment. The normalization of ID1-PCR prod-ct to b-actin revealed no evidence for upregulation

of ID1-specific mRNA upon detachment. To furtheranalyze (post-)translational regulation mechanismsof ID1 expression we detached HaCaT-cells fromtheir growth substratum and incubated them for afurther 8 h in the presence or absence of 10 mgycloheximide/ml, a translation inhibitor. Sheetsere analyzed immunohistologically using mabDpKeID1a 5.21. We found no change of ID1-expres-

ion in the presence of cycloheximide. It was stillound upregulated in the cytoplama and translocated

n the nuclei (data not shown).

DISCUSSION

To analyze the inhibitor of DNA-binding ID1 in thehuman epidermis and in cultured keratinocytes wegenerated and characterized monoclonal anti-ID1 an-tibodies. Immunohistological studies on human normalepidermis and on lesional epidermis of bullous pemphi-goid patients revealed that ID1 is not expressed innormal human epidermis as expected from unpub-lished data of Lyden [26] but is expressed in lesionalepidermis of bullous pemphigoid patients. In the lattercase ID1 was found in the cytoplasm of basal andproximal suprabasal keratinocytes, which—to ourknowledge—provides the first evidence for ID1 expres-sion in vivo under nontumorgenic conditions [27]. Cul-tured nonconfluent and confluent normal human epi-dermal keratinocytes were found to express ID1 in thecytoplasm; after differentiation into a multilayeredsheet ID1 was no longer detectable. However, it wasreexpressed in the multilayered sheet after dispase-mediated detachment and further cultivation for 8 h.In this case ID1 was localized to the nucleus and cyto-plasm.

In the context of epidermal (patho-)physiology ourdata indicate that ID1 may participate in the differen-tiation of the epidermis as well as in the cellular reac-tion to loss of cell matrix contact. ID1 appears to benegatively regulated upon in vitro differentiation ofkeratinocytes, since it was immunohistologically de-tectable in monolayered (Fig. 6) but undetectable inmultilayered epidermal keratinocyte cultures (Fig. 4A)and normal differentiated epidermis of the skin. Thesefindings are in agreement with data from Alani et al.[12], who showed that ID1-transfected keratinocytesare less prone to differentiation than untransfectedkeratinocytes. The effect of ID1-expression in keratin-ocytes thus resembles the effect of ID1 in muscle cellsor B cells. In these cells downregulation of ID1 is dis-cussed as a prerequisite for differentiation [7, 28]. Inview of these findings we suggest that downregulationof ID1 in confluent keratinocytes may influence epider-mal differentiation possibly via the induction of growtharrest [17], which occurs prior to differentiation. In thecontext of the physiologic epidermal differentiationID1 thus may be a key regulator.

ID1 is upregulated upon dyshesion of keratinocytesfrom their growth substratum caused by autoantibod-ies in vivo (bullous pemphigoid (Fig. 3)) or dispasetreatment in vitro (Fig. 4). This upregulation does notseem to be reflected in increased mRNA levels. Fur-thermore the presence of the translation inhibitor cy-cloheximide had no influence on ID1 expression, sug-gesting a posttranslational regulation mechanism.This is consistent with data of Jen et al. [29], Simonson

et al. [30], and Springhorn et al. [21], who already

257ID1 EXPRESSION IN EPIDERMAL KERATINOCYTES

suggested a (post-)translationally regulation of ID pro-teins.

Since we previously demonstrated that dispase-in-duced dyshesion of keratinocyte sheets leads to activa-tion reaction of keratinocytes [14, 18] and in view of ourpresent data in particular that three of five bullouspemphigoid lesions express ID1, we suggest ID1 as anadditional marker of activated keratinocytes [18, 31,32]. Since the analysis of an array of other epidermalautoimmune diseases not associated with cell–matrixdisturbances such as lupus erythematodes and othersrevealed no hints for ID1 expression we speculate that

FIG. 5. (A) Lysates of NHEK and HaCaT sheets directly afterand 8 h after dispase treatment were analyzed for ID1 expressionusing Western blot technique as detailed under Materials and Meth-ods. Mabs used were HDpKeID1a 5.21 (lanes 1–4) and 210.12 (lanes5–8). A 16-kDa band appeared in the NHEK lysate 8 h after dispasetreatment (lanes 2 and 6), whereas directly after dispase treatment(lanes 1 and 5) no ID1 was detectable. HaCaT lysates appeared to beID1-negative directly after (lanes 3 and 7) and 8 h after (lanes 4 and8) dispase treatment. Lane 1, NHEK lysate directly after dispasetreatment; lane 2, NHEK lysate 8 h after dispase treatment; lane 3,HaCaT lysate directly after dispase treatment; lane 4, HaCaT lysate8 h after dispase treatment. Lanes 1–4 were developed with mabHDpKeID1a 5.21. Lane 5, NHEK-lysate directly after dispase treat-ment; lane 6, NHEK-lysate 8 h after dispase treatment; lane 7,HaCaT lysate directly after dispase treatment; lane 8, HaCaT lysate8 h after dispase treatment. Lanes 5–8 were developed with mabHDpKeID1a 10.12. (B) HaCaT lysate directly after (lanes 3, 4, 7, and8) and 8 h after (lanes 1, 2, 5, and 6) dispase treatment weresubjected to immunoprecipitation using the rabbit anti-ID1 serumand normal rabbit IgG. The immunoprecipitate was analyzed viaWestern blot technique using the mabs HDpKeID1a 5.21 (lanes 1–4)and 210.12 (lanes 5–8). ID1 was only detectable in HaCaT lysate 8 hafter dispase treatment (lanes 2 and 5). Lane 1, HaCaT lysate 8 hafter dispase treatment immunoprecipitated with normal rabbit IgG;lane 2, HaCaT lysate 8 h after dispase treatment immunoprecipi-tated with rabbit anti-ID1 immunoglobulins; lane 3, HaCaT lysatedirectly after dispase treatment immunoprecipitated with normalrabbit IgG; lane 4, HaCaT lysate directly after dispase treatmentimmunoprecipitated with rabbit anti-ID1 immunoglobulins; lanes1–4 were developed with HDpKeID1a 5.21. Lane 5, HaCaT lysate 8 hafter dispase treatment immunoprecipitated with normal rabbit IgG;lane 6, HaCaT lysate 8 h after dispase treatment immunoprecipi-tated with rabbit anti-ID1 immunoglobulins; lane 7, HaCaT lysatedirectly after dispase treatment immunoprecipitated with normalrabbit IgG; lane 8, HaCaT lysate directly after dispase treatmentimmunoprecipitated with rabbit anti-ID1 immunoglobulins; lanes5–8 were developed with HDpKeID1a 10.12.

ID1 expression might be triggered via a disturbed cell–

matrix and/or cell–cell interaction. We are thereforeparticularly interested in reepithelialization processesduring wound healing and are currently establishing amouse model to study this matter in more detail. Wecannot rule out, however, that the induction of ID1expression in vitro may be due to the reexposure of thekeratinocytes to the serum in the old medium and thatthe ID1 expression reflects reinduction of proliferationafter growth arrest, a process in which Id proteins havelong been implicated [17]. In favor of this argument itmay be worthwhile to mention that epidermal lesionsin vivo are accompanied by the exudation of plasma/serum proteins, causing upregulation of ID1 in epider-mal cells.

Analyzing our immunohistological data more closelywe found that ID1 is expressed in two different subcel-lular localizations: in the cytoplasm of cultivated mono-layered keratinocytes and lesional keratinocytes inbullous pemphigoid biopsies and in the nucleus of dis-pase detached keratinocytes. We therefore assumethat the function of ID1 may differ depending on itssubcellular location. Taking into consideration that (1)the phosphorylation status of ID1 may modulate theinteraction with its target proteins [33, 34] and (2) thenuclear localization of IDs seem to critically depend onthe presence of the ID interaction partners [24], wespeculate that dispase treatment either changes phos-phorylation status of ID1 in keratinocytes, allowinginteraction with an already present interaction part-ner, or triggers the production of an ID1 interactionpartner that—via a chaperone mechanism [24]—lo-cates ID1 into the nucleus. Our immunohistologicalstainings showing ID1 in the cytoplasm of keratino-cytes may therefore either indicate the presence offunctional ID1 lacking its interaction partner or non-functional phosphorylated ID1 or even via the ubi-quitin–proteasome pathway degraded ID1 [25].

In view of the finding that ID1 is expressed in dis-pase detached keratinoctes, we expected a nuclear andcytoplasmic location of ID1 in lesional keratinocytes ofbullous pemphigoid. However, we found ID1 only in thecytoplasm of these keratinocytes (Fig. 3). This may beexplained by the short half-life of ID1. Although it hasbeen shown that the half life of complexed ID1 is longerthan that of uncomplexed ID1, it does not exceed sev-eral h [25]. We have been unable to analyze nuclearlocalization of ID1 in early bullous pemphigoid lesionssince no biopsies of early bullous pemphigoid lesions(,48 h) were available. Alternatively the difference inthe nuclear ID1 expression might be due to differentagents inducing keratinocyte activation: on the onehand, dispase, which cleaves extracellular matrix pro-teins [35] and the hemidesmosome [36], and on theother hand, autoantibodies that bind to and disturb thefunction of the hemidesmosome [37]. The different

mechanisms for activating keratinocytes might be re-

1

1

1

1

1

1

1

1

1

258 SCHAEFER ET AL.

flected in variable cellular reactions and protein ex-pression patterns [32], in this case in differences in ID1expression and localization.

Finally the putative function of ID1 in epidermalkeratinocytes remains to be adressed. Several aspectshave to be taken into account: First it was shown byDesprez et al. [38] that ID1-expression destabilizescell–cell contacts in mammary epithelial cells. Duringepidermal wound healing keratinocytes from the bor-der of the lesion leave the epidermal cell layer andreepithelialize the defect. These keratinocytes are saidto change their phenotype from the sessile into themobile keratinocyte [39]. In line with this assumptionare data from Lin et al. [27], showing that ID1 expres-sion in breast cancers are important indicators of theaggressive and invasive phenotype. Under these con-ditions ID1 may augment the dissociation of singletumor cells from the primary tumor, leading to metas-tasis formation. Along these lines we suggest that ID1expression might facilitate cellular reactions that arenecessary to induce the mobile phenotype in epithelialcells in general and in keratinocytes in particular.

In another report ID1 was found to be a regulator ofcell mitosis by increasing cell proliferation [40]. ID1induction of proliferation in keratinocytes of bullouspemphigoid might therefore supply the amount of ker-atinocytes needed for closure of epidermal defects.

Third, ID1 inhibits cell differentiation [7, 28], aneffect that is generally observed in healing epidermalwounds. Taken together we therefore hypothesize thatID1 may be a key regulator of epidermal wound heal-ing processes facilitating reepithelialization, whilestimulating proliferation and inhibiting differentiationof keratinocytes.

According to this scenario we suggest ID1 as animportant player in epidermal (patho-)physiology andmarker of keratinocyte activation. After epidermal in-jury—in our case loss of cell–matrix contact—ID1 isupregulated, thus facilitating the change from thesessile to the mobile keratinocyte and stimulating ker-atinocyte proliferation both of which are required forefficient reepithelialization. After reepithelializationhas been acchieved, ID1 is downregulated, allowingepidermal cell differentiation into a multilayer.

This work was supported in part by the Forschungskommission ofthe University of Heidelberg (No. 250/2000) and the Deutsche For-schungsgemeinschaft (SCHA 862/2-1 and Kr 931/3-3). We thankChristine Nitsch and Silke Gull for their expert work in generatingand characterizing the monoclonal antibodies.

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Received August 30, 2000Revised version received February 28, 2001