Herlitz junctional epidermolysis bullosa
keratinocytes display heterogeneous defects of
nicein/kalinin gene expression.
C Baudoin, … , G Meneguzzi, J P Ortonne
J Clin Invest. 1994;93(2):862-869. https://doi.org/10.1172/JCI117041.
Previous studies have correlated the Herlitz junctional epidermolysis bullosa (H-JEB) to an altered expression of the basement membrane component nicein/kalinin. This
heterotrimeric glycoprotein appears to be present in H-JEB tissues in an abnormal form, because a number of antibodies specific to the protein either do not react with or weakly stain the epidermal basement membranes of most of the patients. With cDNA probes encoding each subunit of nicein and polyclonal antibodies raised against bacterial fusion polypeptides corresponding to the individual chains of the protein, we have molecularly analyzed the expression of nicein in H-JEB tissues and cultured keratinocytes. By
immunohistochemistry, Northern blot, and protein analysis, we show a defective synthesis of one of the nicein subunits in six cases of H-JEB from five different consanguineous families. In two patients, the disease correlates with an impaired synthesis of the nicein B2 (nic B2) chain, in three others with that of the B1 (nic B1) chain, and in a sixth patient with that of the heavy A (nic A) chain. In this report, we thus demonstrate that H-JEB is a
genetically heterogeneous disease and we provide strong evidence that the genes of nicein are the candidates for this genodermatosis.
Research Article
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Herlitz
Junctional
Epidermolysis
Bullosa
Keratinocytes
Display
Heterogeneous
Defects of
Nicein/Kalinin
Gene
Expression
Christian Baudoin,*Corinne Miquel,*Claudine Blanchet-Bardon,t Claudio Gambini,' GuerrinoMeneguzzi,*
andJean-Paul Ortonne*
*INSERM U385, UFR de Medecine, 06107Nice; tCliniquedes Maladies Cutaneeset Unite de Recherche,
DiagnosticAntenatalenDermatologie, H6pitalSaint-Louis, 75 Paris, France;and§Serviziod'Istologia,
OspedaliGalliera, 16128 Genova, Italy
Abstract
Previous studieshave correlated the Herlitzjunctional epider-molysis bullosa (H-JEB)toan altered expression of the base-ment membrane component nicein/kalinin. This heterotri-mericglycoproteinappearsto bepresentin H-JEB tissues in anabnormal form, becauseanumber of antibodiesspecificto theproteineither donotreact withorweaklystain the
epider-malbasement membranes ofmostofthepatients.WithcDNA probes encoding eachsubunitof nicein and polyclonal antibod-ies raised against bacterialfusion polypeptidescorresponding
to the individual chains of the protein, we have molecularly analyzed theexpression ofnicein in H-JEB tissues and cul-tured keratinocytes. By immunohistochemistry,Northernblot,
andprotein analysis,weshowadefectivesynthesisofoneof the
nicein subunitsinsixcasesofH-JEB from five different con-sanguineous families. In two patients,the disease correlates withanimpairedsynthesisof the nicein B2(nicB2) chain,in three otherswiththatofthe B1 (nic Bl) chain,and in a sixth
patient withthatoftheheavyA(nicA)chain.Inthis report,we thus demonstratethat H-JEBis agenetically heterogeneous diseaseand weprovidestrongevidence thatthe genesof nicein
are thecandidates for this genodermatosis. (J. Clin. Invest.
93:862-869.)Key words:nicein/kalinin*basement membrane
*junctional epidermolysisbullosa*dermal-epidermal junction-hemidesmosomes
Introduction
Hereditaryepidermolysisbullosa(EB)'isa groupof blistering skindiseases characterizedbytissueseparationwithinthe
der-mal-epidermal basement membrane zone (1, 2). In the sim-plex forms ofEB,intraepidermal tissueseparationoccursat the
level of basal keratinocytes( 1). The different clinical manifes-tations ofEBsimplex havebeen recently attributedto muta-tionson thegenesencodingkeratins 5 and 14,which are
ex-AddresscorrespondencetoDr. Jean-Paul Ortonne, INSERM U385,
UFR deMedecine, AvenuedeVallombrose, 06107 Nice Cedex 02,
France.
Receivedfor publication30August 1993andinrevisedform3
No-vember 1993.
1.Abbreviations used in thispaper:EB,epidermolysis bullosa; H-JEB,
Herlitzjunctionalepidermolysisbullosa; HD,hemidesmosome;nic A,
niceinAchain; nicB1,niceinB1chain; nicB2, nicein B2 chain; pAb,
polyclonalantibody.
pressedby
basal keratinocytes
(3-5).Inthedystrophicscarring
formsofEB, the cleavageoccurswithin thepapillarydermis, below the basal laminaatthe level of theanchoring
fibrils
(1). Several linesof evidence identifiedtype VIIcollagen, the main component ofanchoring fibrils,
asthe candidategenefor dys-trophic EB (6, 7). Subsequent genetic analysis of affected kindreds and theidentificationof point mutations inthe colla-gen VII DNA sequences strongly support the involvementof thegenein thepathology(8-10). Thejunctionalforms ofEB(JEB) result from tissueseparationwithin the lamina lucida of thedermal-epidermal basement membrane. Several
varieties
of JEBhavebeendescribed
(1).
Thegeneralizedform,ortheHerlitz's variant
(H-JEB), presents the most severe clinical manifestationswith widespreadblistering
of skinandmucousmembranes. In most cases, H-JEB is fatal early in life. The
non-Herlitz
variants
arecharacterized
by continuousblistering from birthorearly infancy that doesnotinterferewith normal development and life span. All formsofJEB are autosomal recessive.Ultrastructuralstudies of JEB skin havedemonstrated ab-normalitiesinhemidesmosome (HD)shapeandnumber(
(11
). HD arecellular structures that appearaselectron microscope thickenings making connection with intermediate filaments(12, 13). They arelocatedat the interface of basal epithelial cells with theunderlying dermis,andarepresumedtofunction
as part ofan epithelial cell-substratum attachment device
(14).Considerableprogresshasrecentlybeen madein unravel-ing themolecularcomposition ofHD
(12).
Domains ofthebullous
pemphigoid
antigens1(BPAGl
)and2(BPA G2) (15, 16) and thoseofa200-kDpolypeptide( 17)residein the hemi-desmosomal plaquetowhich keratin bundles attach. An addi-tional high molecular mass component (HD1)
is locatedonthe
cytoplasmic
side ofHD( 18).Integrin
a6/34
is also found in theHD( 19-22),and thecytoplasmic domainsof theproteinmayplayarole in thejunctional connectionsof hemidesmoso-mal structurestothecytoskeleton ( 12).
The
bridge
between theHDandthe lamina densaregion ofthe basement membrane is provided by the
anchoring
fila-ments( 12). Noncollagenousproteins immunolocalizingwith
these structures have beenidentified. They
comprise
nicein/
BM-600 (23, 24)andkalinin (25), whichhave been
demon-stratedtobethe sameprotein (26).Nicein/kalininisa
hetero-trimeric
protein containingthreenonidentical subunits, cova-lently linked by disulfide bonds (24, 25). The molecularmassesofthe threechainsasreported bytwodifferent groups
are,respectively, 100/105 kDforthelightest subunit(nicB2), 125/140kD for the second chain(nic
Bl),
and 150/165 kDfortheheaviest subunit(nicA).Ithas beendocumentedthat
maturation of nicein/kalinin occurs extracellularly in two
steps from ahighmolecularmassprecursor(27). cDNA en-codingforthe threesubunitswererecently isolated,andtheir
862 Baudoinetal. J.Clin.Invest.
C TheAmericanSocietyfor ClinicalInvestigation,Inc.
sequences revealed that nicein is alaminin-like protein (28-30). nic B2 is homologoustothe recently reported laminin-like chain B2t (31). Laminin and nicein both display a cross-shaped conformation, nicein presenting the shortarmsof the crossreducedinsize (27). The novel laminin variant, K-lami-nincontaining 220-kD B1and 2 10-kD B2 laminin chains, as well as a 1 90-kD novel chain, is also associated with these
he-midesmosomal structures (32). Less well-characterized an-choring filament componentsaretheprotein epiligrin and the 19-DEJ- 1 (33, 34). Epiligrin antibodies precipitate peptides withelectrophoreticmigrations similar to those ofboth nicein / kalinin and K-laminin (35). The molecular mass of the 19-DEJ- 1 antigenis unknown(34).
No large scale abnormalities in the BPA G1 gene were
foundbySouthern analysis of H-JEB genomic DNA (36).
Im-munohistological studies have demonstratedthat a6f34 (37)is normally expressedbyH-JEBbasal keratinocytes. a6f4 never-thelessappears more defocalized at central basal areas of the
cells(38). On thecontrary, the expression ofnicein/kalinin is
dramatically altered(39-42). Antibodies directed against
ni-cein donot reactwith the epidermalbasement membranesof
most ofH-JEB patients and abnormal staining can also be
observedinthenon-H-JEBpatients (40, 41 ). Indirect immuno-fluorescence studies ofH-JEBskinusingpolyclonal antibodies (pAbs)tothe individual purified subunits ofnicein suggest that
the protein is present butstructurally altered(43). However,
because of the cross-reactivity between pAbsdirectedagainst the nicB2 andnicAsubunits,the causeunderlying the unde-tectabilityof nicein in H-JEBskin was notdetermined.
The availability ofcDNAs encoding for the three nicein
subunitsallowed the production ofpAbs directed against
fu-sionproteinscorrespondingtothenicB2andnic A chainsof human nicein. These antibodies and the corresponding nicein cDNA probes have been used to follow thesynthesis ofthe different chains of nicein in skin and culturedkeratinocytesof
H-JEBpatients. Methods
Tissues. Skinbiopsieswerefrom six H-JEBpatientsfromfive different families:twofrom 20-21-wk-oldfetuses(Gf,F)and four from
new-borns(L, Gb,B,S).In allcases,H-JEBwasdiagnosedaccordingtothe revised criteria forsubtypingEB(1).The levelof cleavagein all le-sional skinsampleswasconfirmedaccordingtoimmunofluorescence mappingofVLA-6, laminin,and typeIVcollagen.Transmission
elec-tronmicroscopyshowedadermo-epidermalsplitin the lamina lucida. Allsamplespresentedabnormal HD. The skinbiopsiesdidnot react
with GB3or 19-DEJ-1 mAbs(44).Controlswerenormalfetal skins from 18-24-wk-old fetuses and from adulthealthyvolunteers.
Antibodies.Forthecharacterizationof basement membrane
com-ponents, thefollowingmAbsorpAbswereused: mAb GB3
(hybrid-omaculturemedium; working dilution, 1:2) (45)rabbitpAb
anti-hu-man collagen IV (working dilution, 1:2; Institut Pasteur, Paris, France), rabbit pAb anti-human collagen VII (working dilution, 1:10),goat pAb anti-mouse laminin (working dilution, 1:50; Sigma Chemical Co., St. Louis, MO) mAb anti-EBA antigen (working dilu-tion, 1:50), mAbLH 7:2(working dilution, :10),mAb 19-DEJ 1 (working dilution, 1:50), mAb GB36 (working dilution, 1:2). Three
serafrompatientswith herpesgestationis(working dilution, 1:2-1:5) and five from patients with bullous pemphigoid (working dilution,
1:50-1:00)were also used.
Theantinicein pAbs SE 144 and SE 85 were raised against fusion proteins derived from cDNAs encoding the central domain of the nic B2 subunit and the COOH-terminal domain of the nic A subunit, respectively (27, 30). The pAba125 specificallyrecognizes the nic
Bl
subunit of nicein (43). The mAb K 140 specific to the nic B 1 subunit of nicein/kalinin (31 ) and themAbBM 165 specific to the nicAchain of nicein/kalinin, its 200-kD precursor (25), and to the 190-kD subunit of K-laminin have also been used (31). Nonimmune sera (normal human sera andpreimmunizedrabbit sera or culture medium of
P3-NSl-Ag4-1 (NS 1 cell line) were used as control.
Cellculture. Splitthicknessepidermis was separated from dermis by dispase treatment at370C.Keratinocytes were dissociated in trypsin 0.25% at370C, and plated on a feeder of irradiated 3T3 cells (ICN, Costa Mesa, CA) (46). Keratinocytes were grown in a 1:1 mixture of DMEM and Ham's F12 (GIBCO BRL, Eragny, France) containing 10% FCS, 1 mM sodium pyruvate, 2 mML-glutamine, 10,g/mlof penicillin and streptomycin, 10 ng/ml EGF,100pMcholeratoxin, 400 ng/ml hydrocortisone, 5 ,ug/ml transferrin, 180MMadenine, and 20 pM T3 (47). H-JEB keratinocytes were expanded after gentle dissocia-tion in 0.05% trypsin, 0.02% EDTA.
Radioimmunoprecipitation assays. Primary keratinocytes (0.6
X 106 cell/well) were seeded in9-cm2-wellplates and allowed to attach for 12 h. Cells were then labeled for 24 h in 1.5mlof methionine- and
cysteine-deficientculture medium(GIBCOBRL) supplemented with 100,Ci/ml [35S]methionineand[35S]cysteine (Amersham Interna-tional, Amersham, England). Culture medium was centrifuged at
12,000 rpm to remove cells and debris, and TCA-precipitable radioac-tivity was counted. Preclearing was performed by adding aliquots of each sample (- 5 x 106TCA-precipitablecpm) toapellet of protein A-Sepharose 4B beads (Pharmacia SA, Uppsala, Sweden)
precom-plexed with either rabbit anti-mouse IgG (Dakopatt, Copenhagen, Denmark) or preimmune rabbit serum, respectively, for further use with mAbs and pAbs. The reaction mixtures were incubated for 1 h at 4°C under rotatory agitation. Precleared aliquots were then centri-fuged, and supernatant incubated with mAb K 140(nicBI) atafinal dilution of 1:100orwith SE 144(nic B2) atafinal dilution of 1:40 overnightat 4°Cunder rotatory agitation. For control conditions a
preimmune rabbit serum was used at a final dilution of 1:40. Samples were then incubated with pellets of Sepharose 4B beads for1 h at4°C.
After centrifugation the supernatant was removed and the pellet washed once with cold Tris-buffered saline,0.1% NP-40, briefly vor-texed, then extensively washed four times with radioimmunoprecipita-tionbuffer (RIPA buffer: 10mMTris-HCl, pH 7.4, 150mMNaCl, 2
mM EDTA, 250MM PMSF, 1 mM n-ethylmaleimide, 2 mM
L-me-thionine, 2 mML-cysteine,0.3% NP-40, 0.05% Triton X-100, 0.3% sodium deoxycholate, 0.1%BSA) containing 0.1%SDS (31),and then with RIPA buffer containing 0.5 M NaCl, and rinsed once with TBS. Immunoprecipitated materials were then analyzed on a 6% SDS-PAGE gelelectrophoresis (48).
Northernblotanalysis.Total RNA was prepared from H-JEB and normal cultured keratinocytes according to the method of
Schom-czyskiandSacchi(49).RNAwassubjected to electrophoresis on 1.2%
denaturing agarose gels containing 1.2 M formaldehyde and trans-ferredontonylon membrane (Hybond N; Amersham International). Membranesweresubsequently hybridized at high stringency with 32P-labeled cDNA probes corresponding to the different chains of nicein and thenexposed 16honHyperfilm MP (Amersham International) withintensifying screens.
Theradiolabeled cDNA probes PCR 1.3 (27), KAL-5.5C (28), andNA 1 (29)wereused to detect the mRNA for the nic B2, nic B 1, andnicAchains, respectively.
Results
Immunohistochemicaldetectionofniceinchainsin normaland H-JEB tissues. Immunofluorescence experiments were per-formedonsamples ofhuman
epidermis
usingpAbsspecifically
directedagainsteachsinglechain of nicein
(Fig.
1and TableI).
I
864 Baudoin etal.
skin, all the antibodies labeled theepidermal basement
mem-brane (Fig. 1,A-C).In mostbasal keratinocytes, pAb Pa 125 (nic Bl) (Fig. 1 B) and pAb SE 85 (nic A) (Fig. 1 A) gave a cytoplasmic staining, whereas pAb SE 144 (nic B2) reacted morefaintly with the cytoplasm of only some of the basal cells
(Fig. 1 C). Results obtained with patients Gb and Gfwere
consistentwith observationsmadeonpatientF. Patients Aand Lhadsimilar patterns of reactivityto theantinicein antibodies.
Patient S presented adistinct immunological reactivity.
Repre-sentative resultsareshown inFig. 1, D-I.
Inpatients Gb, Gf, and F,the pAb Pa 125 (nic B 1 ) failed to react with both the dermal-epidermaljunction and the
cyto-plasm of epidermal keratinocytes,while staining with the pAbs
SE144(nic B2) andSE 85 (nic A) was indistinguishable from
that of normal controls, with the exception ofthecytoplasmic staining, whichwas markedly stronger(Fig. 1, D and F). This resultsuggests animpairmentofthe synthesis and / or the
secre-tion ofthe nicB 1 chain, since the otherchains of niceinare
apparently normally expressed. Tissue from patientsA and L
exposedtopAb SE 144(nic B2)showed noimmunoreactivity (Fig. 1 !). pAb Pa 125(nicB1)alsodid not label the epidermal
basement membrane of these patients,but presenceofthenic
B 1 subunitwasdetected in isolatedbasalkeratinocytesin
pa-tientA(Fig. 1H).Inbothpatients, the expression ofthe heavy
chain(nic A) of niceinwascomparabletothat ofhealthy
con-trols(Fig. 1 G). Clearly, in thesetwo casesonlytheexpression ofthe nicB2chainwascompletely absent.Both the pAbs SE 85
(nicA)(Fig. 1 J) and Pa150(nic A) (data not shown) did not reveal anyspecific staining; a cytoplasmic reactivity was
ob-tained with pAbPa 125 (nic Bl)(Fig. 1 K) and pAb SE 144
(nic B2)(Fig. 1 L), which indicatesalowrateof synthesis of thelightchains of nicein. Thus, in thiscase,only theexpression of the nicA chain is undetectable. All otherantibodies tested
(see Methods) stained theepidermalbasement membrane
nor-mally (datanotshown).
Expression ofnicein mRNA in H-JEBkeratinocytes. To
check whether the altered expression ofnicein subunits
de-tectedby immunofluorescence reflected possible structural
al-terationsof nicein chainsorabsenceof nicein chains, the
ex-pression of niceingenes wasexaminedbyNorthern blot
analy-sis ofmRNApurified from primary cultures of keratinocytes obtainedfrom the sixH-JEBpatients.Total mRNA was
frac-tionated byagarosegelelectrophoresis, blottedonto nitrocellu-lose, and subsequently hybridizedtothenick-translatednicein
cDNAprobes.
Consistent with the immunofluorescence observations in patientsGband F,theprobespecificfor nicB 1 chain failedto
detect thecorrespondingmRNA, whereas the nicB2chainand
nicA chainmRNAwere normally expressed (Fig. 2).
How-Table I. Iminunofluorescence Data
Nicein chain
nicA nic Bi nic B2
Patients EC IC EC IC EC IC
Gf and Gb ++ ++ - - ++ ++
F ++ ++ - - ++ ++
A ++ + _ +*
L ++ +* - - _ _
S - - - - ++
Controls ++ + ++ +* ++ +*
Scoredimmunofluorescence withpAbsSE 85 (nicA), Pa 125(nic
BI),andSE 144(nicB2)in H-JEBskin and in normal controls. Absence of fluorescence (-), detectable levels ofstaining (+),and
strongfluorescence (++)areindicated. EC, extracellularstaining (labeling of thedermo-epidermal junction). IC, cytoplasmicstaining ofepidermalbasalkeratinocytes. *Stainingof scatteredepidermal keratinocytes.
ever,the nicB 1 chainmRNAcould be detectedas avery faint
band of4 kb in culturedkeratinocytesfromanaffected fetus (Gf)oftheGfamily(Fig. 2B). This suggests that its lack of
detection in the otherpatientoftheGfamily (Gb)wasdueto a
strong decreaseintranscriptionor mRNAstabilityrather than
toatotalabsence.These results notonly confirmedthe immu-nofluorescence observations, indicatingapossible altered
ex-pression of the nicB 1 chain, but they further demonstratedan
impairment of transcription or/and an instability of the
mRNA.
Inthe caseof patientAandconsistentwith
immunofluores-cencedata, themessengerscorrespondingtothe chainsnic B 1
and nicAwerenormallyexpressedasstrongbands of4and6 kb, respectively, while the nic B2 mRNA were undetectable (Fig. 2 C).Inpatient L,whereantibodiestothenicB2and nic
B 1 chains gave no immunoreactivity, only the nic B2chain
mRNA was not detected. Thus, Northern blot analysis and
morphologic observations both demonstrate that thesynthesis ofthenicB2chainisspecifically impairedin these twocases.
In patient S, the mRNA corresponding to all the three chains of nicein were present, but the mRNA for the nic A chainwasgreatlyreduced(Fig.2A), whereas theexpressionof the mRNA for the othertwo chains appeared normally ex-pressed. No appreciable changes in the size of the different
mRNAwereseen.
Synthesis ofniceinchains.Innormalhumankeratinocytes (Fig. 3, lanes2 and3), mAbK 140(nicBl ) and pAb SE 144
Figure1.Immunofluorescence analysis withthe polyclonal SE 85 (anti-nic A), SE 144 (anti-nic B2), and Pa 125 (anti-nic B1)of healthy
(A-C) andH-JEBfetal skin (D-L).(A-C) Control fetalskin. All theantibodieslabel the epidermal basement membrane. pAb SE 85 (nic A)
alsostains basal cells (A).pAbs Pa125(nicBI) (B) and SE 144 (nic B2) (C) give a faint cytoplasmic staining restricted to isolated keratinocytes.
(D-F) PatientGf.pAb SE 85 (nicA)givesthetypical linearpatternof fluorescence at the dermal epidermal junction; a strong cytoplasmic
staining of basal keratinocytes is also observed(D). pAb Pa 125 (nic B1)does not label the epidermal basement membrane (E). With pAb SE 144(nic B2), the staining of the dermal epidermal junction iscomparable to that obtained in controls although with a stronger cytoplasmic
reactionwithbasalkeratinocytes. (G-I) PatientA. pAb SE 85 (nic A) reacts as in control skin (G). With Pa 125 (nicBl),theepidermal base-mentmembraneisnotreactive, but scattered basal keratinocytesshow acytoplasmic staining(H). No reactivity is detected with pAb SE 144
(nic B2) (I). (J-L) Patient S.Noneof thethree pAbs, SE 85 (J), Pa 125 (nicBl) (K), and SE 144 (nic B2) (L), stain the dermal-epidermal
junction. pAbsPa125 (nicBI) (K)andpAb SE144(nicB2) (L) decorate the cytoplasm of isolated basal keratinocytes. With pAb SE 144 (nic
K Gf A Gb L K F7 S K K Gf A Gb L K F S K Gf A G(b L K F S Figure 2. Northern blot analysis of
Kb Kb Kb human normal (K) and H-JEB
ker-AABB CC atinocytesof total RNAfrompurifiedsixpatients.fromprimary10 g
j== = ft^cultures ofkeratinocyteswas
sub-_t
-Aa
i
jectedtoelectrophoresisonagarose6. _ _ * 4b.
_5.3-
^ * s _ gels,blotted to a nitrocellulose sheet,V andsuccessivelyprobedwith
3P-la-beled cDNAs for thenicein chains nic A (A), nic BI (B), and nic B2
NICA NICB1 NIC B2
(C),
respectively. (A) In patient Sthe mRNAfor the nic A chaingives asignal reduced in intensity. (B) In patients Gb and Gf, the mRNA specific for nicBl is undetectable or its amount strongly reduced. (C) In patientsAand L the mRNAfor the nic B2 chain is not detected.
(nic B2) immunoprecipitate the mature form of nicein present inconditioned culture media, as documented by the detection
of the three subunits of the proteinat the molecular massesof
105, 140, and 160 kD, respectively, which approximate those previously reported for the mature forms of nicein and kalinin. Afaint band at 150 kD was also present, consistent with unpro-cessed B2chainpreviously reported (27). An additional band at250 kD was visible in all the samples including controls. The bandscorrespondingto thenicein subunitswere not observed whenradioimmunoprecipitationwas performed with the con-trolpreimmune serum (Fig. 3, lane 1), while the band at 250 kDwasclearlydetectableincontrol samplefromH-JEB
kera-tinocytes fromall thefive patients studied(patients L and Gf;
Fig. 3, lanes4and 7; not shown forthe three otherpatients)
and, faintly, in normal keratinocytes. It was thus concluded
thatthe250-kDbandis nonspecificand presumably be due to fibronectin.
InpatientL, mAb K 140immunoprecipitated four
polypep-tides of 200, 185, 160,and 138kD.Consistent withtheresults of Northern blot analysis, the 105-kD nic B2 chain was not detectable. In parallel radioimmunoprecipitation assays
per-formed with pAb SE 144, which recognizesthenic B2subunit,
nospecific band wasimmunoprecipitated (Fig. 3, lane6). A similarimmunoprecipitation pattern was observed in
experi-mentswithculturedkeratinocytes fromthepatientA(datanot
shown). These data definitely confirmthat in these patients synthesisofnicB2chainisimpaired.
InpatientGf, themAb K140specifictothe nicB 1subunit,
whose mRNAs are undetectableinkeratinocytesfrom this
indi-K
L
I I I I
1 2 3 4 5 6
_~~~_ _
NIC
A-NIC
Bi-NIC
B2-vidual,failedtoimmunoprecipitateanyspecificbands(Fig. 3,
lane8). Conversely, with pAb SE 144(nic B2),at least nine bands were observed:major bandsof230, 200, 160, 155, 105, 85, and 80kDand twofaint bands at 175 and 125 kD(Fig. 3).
Onlythe bandcorrespondingtothe nic B 1 chainat140 kDwas
not detectable.Asimilar pattern was observed withpatientF
(data not shown). The involvement of the Nic B1 wasthus clearly demonstrated.
Inpatient S,where no immunoreactivitywasobservedat
the dermal-epidermal junction, whateverthe antibody used,
whilepAb SE 144 (nic B2) andPa 125 (nic BI) detecteda
cytoplasmic staininginbasalkeratinocytes (Fig. 1, J-L),both
pAb 144(nic B2) and mAb 140(nicBI) immunoprecipitated the three nicein chains. As in the other H-JEBpatients, the
bandscorrespondingtothe niceinchains were reduced in
in-tensity, comparedwith thecounterpartsfromhealthydonors.
Inall casesbands similartothosefoundinthe culture medium were seen inthe cell extractfromthe H-JEBkeratinocytes (not shown).
Discussion
We previously reported that most H-JEB patients present a
widespread defect of nicein. Thiswasdemonstrated by thelack
ofreactivity ofH-JEBskin andepitheliato the mAb GB3(41 ). Since nicein couldnotbeimmunoprecipitated from cultured
H-JEB keratinocytes with these antibodies, it was suggested
that the abnormalexpressionof theproteincould result from
intrinsic defects of H-JEBepithelial cells (41 ). The molecular
Figure 3. Immunoprecipitationof radiolabeled nicein from culture medium conditioned by human normal (K) and H-JEB
keratino-G F cytes. Cultures of normal foreskin (lanes 1-3) and H-JEB keratino-cytes frompatientsL(lanes4-6)andGf(lanes7-9)werelabeledfor 7 8 9 24 h with[35S]methionineand
[35S]cysteine.
Mediumwasimmuno-kD precipitatedwith mAbK 140(NICBJ)and(lanes2, 5,and8), pAb
kD
SE 144(NIC B2) (lanes 3,6, and9),orwithpreimmunerabbit U
^^ serum(lanes 1, 4,and 7).Immunoprecipitated proteinswere
sub-*- -200 mittedtoSDS-PAGE. Incontrols,bandscorrespondingtothe chains
-170 of nicein are clearly detected (lanes 2 and 3). In patient L, reaction - . w with mAb K 140 doesnotprecipitatethe 105-kD subunit(NIC B2)
b-116 of nicein(lane 5),andnoimmunoprecipitationband is detected with - -100 pAb SE144,which isspecifictothe nic B2 chain(lane 6).Inpatient
Gf,nobandcorrespondingtothenic B I chain isimmunoprecipitated -69 by the pAb SE 144 (lane 9). No nicein chain is recognized by the
anti-nic B1 mAbK 140. The molecularmassesof themigration markersareindicated.
Table II. Summary ofImmunofluorescence Data, mRNA, andIn Vitro NiceinChainsSynthesis
nicA nic BI nic B2
Protein mRNA Protein mRNA Protein mRNA Patients IF RIP NB IF RIP NB IF RIP NB
Gf EC.IC + + - - + EC.IC + + Gb EC.IC ND + - ND - EC.IC ND +
F EC.IC + + - - - EC.IC + +
A EC.IC + + IC + + - -
-L EC.IC + + - + + - -
-S - + + IC + + IC + +
IF, immunofluorescence (-, absent staining; EC, extracellular stain-ing; IC, intracellular staining). RIP, radioimmunoprecipitation (+, detected; -, undetectable). NB, Northern blot (+, detected; -, unde-tectable). ND, not done.
basis of such an abnormality remained unresolved. Using cDNAprobes encoding each subunit of nicein and polyclonal antibodies raised against bacterial fusion proteins correspond-ingtoeachindividual chain of nicein,wehavefurtheranalyzed
sixcasesofH-JEBfrom five different families.
Informationobtainedfrom immunofluorescence studies of involved skin, radioimmunoprecipitation of incubation me-dium, and extracts of cultured H-JEB keratinocytes, and Northern blotanalysis, consistently demonstratethat inthree patients the disorder correlates with animpaired synthesis of the nicB I chain,intwootherswith that of the nicB2,and ina sixthpatientwith thatof the nic A(Table II). It canthus be
concluded that in H-JEB theimpaired expressionofa mature nicein isconsequent to adefectivesynthesisofoneofits sub-units and that thedermatosisisgeneticallyheterogeneous. This finding isnotunexpected consideringthefactthatnicein
com-prisesthreedifferent subunitssynthesizedby distinctgenes. Inthe caseof patientswhere the absenceofoneof thelight subunits of nicein was documented by immunofluorescence and Northern blotanalysis, immunoprecipitation of
keratino-cyteculturemedium always confirmed the absence ofsynthesis ofthepolypeptide,while secreted forms ofproteinswith
electro-phoretic migration corresponding to the other two subunits
were always identified. Other aberrant nicein proteins were
also specifically recognized by the antinicein antibodies,but
thecorresponding bandspresented abnormal electrophoretic migration patterns, different from those expected for nicein subunits.In one case(patient S),thepathologywasassociated with a lackof immunoreactivityto the anti-nicAantibody. This correlated withadecreasein the steady statelevel ofthe
corresponding mRNA. Immunoprecipitation of culture
me-dium conditioned by thekeratinocytesofthispatientwith anti-bodiestothelightchains of niceindetectedlargeamountsof
the three chains of nicein. It is thus clear that all the three
chainsareproducedandsecretedas acomplex in this patient.
Because theapparentmolecularmassofeachsubunit is
consis-tentwithwhatisexpected, conditionsmayexist invivo, but not inculture, that accountforinstabilityordegradationofthe
nicAchainleadingtoundetectabilityin theskin.
Our results show noappreciablechangesin thesizeof
ni-ceinmRNAsinanyofthe H-JEBfamilies.Inpatientsaffected
in the expression of one of the light chains of nicein, the amountofthecorrespondingmessenger wasdramaticallylow. Whether the abnormalconcentrationofsuch mRNAs is due to adecrease in the synthesis or to a higher decay rate has not been
determined.Among thereasonsthat could lead to such a
situa-tion,one should consider the presence ofmutationsinthe pro-moter regions of the affected gene, a decreased stability of the
transcript due to mutations affecting splicing sites, nonsense
mutationsoccurring before the penultimate exon (50), or
mu-tations/alterations ofregulatory genes ( 51 ).
Niceinis alaminin-likemolecule. It has been demonstrated from in vitro analysis of purified fragments that laminin assem-blyinvolvestheformationof a stable coiled-coil Bl-B2 inter-mediate to which the A chain subsequently interacts to form a morestable triplecoiled-coilmolecule (52). It has not yet been
determined iftheassemblyof nicein and laminin areanalogous
processes, but the finding that a complex formed by a heavy
chainand alight chain of nicein areimmunoprecipitatedfrom theculturemediumof H-JEB keratinocytes suggests that, con-trary to the modelproposed for laminin, in the case of nicein
formation ofa Bl-B2-like dimer, it is not a prerequisite for extracellular secretion. However, from immunoprecipitation
data, the presenceofatruncated third chain of nicein in H-JEB
cellscannot beformallyexcluded, but,consideringthe results
ofthe Northern blot analysis, this possibility seems unlikely. Onthe other hand, a release of a dimer in the culture medium consequent to cell lysis cannot be ruled out.
The nic A chain seems to interactindifferentlywith either
nicB 1 or nic B2, because in patients L and Gf this chain ap-pears assembled either withthe nic B 1 chain or the nic B2
chain, respectively. A complex missing a light chain seems, nevertheless, unstable. In fact, the amount ofimmunoreactive proteindetected at thedermal-epidermaljunction of biopsies
frompatients A, L, Gf, and F is exceedingly lowcomparedwith normal controls. However, the amount of nicein chains
im-munoprecipitated from keratinocyte medium conditionedby
keratinocytes of these patientswas almostcomparabletothat
of normal keratinocytes. Moreover, adegradation of the het-erodimers is suggested by theimmunoprecipitationofpeptides
withelectrophoretic migrationsdifferentfrom those found in medium conditioned by normal cells. For instance,the cells from patients Gfand Lsynthesizetwo polypeptides withan
apparent molecularmassof175/ 180 and 200kD,whichare sizescomparable tothose of the precursor of the A chain of nicein (31). The relative abundance of these two proteins could beexplained byanimpairmentof the maturation pro-cessand/oraninefficientexcretion,eitherasmonomers or as dimeric complexesinvolvingthe anotherchainof nicein. This
would also explain the presence ofthe intracellular
fluores-cencefound in H-JEBkeratinocytes usingantibodiestonicB I
and nicA.
These studies clearly demonstrate the involvement of ni-cein in thepathogenesis ofH-JEB and support the idea that genesfor niceinarethe candidate for this disorder. However,
involvementof other molecules intheetiologyofthedisease
cannot beexcluded, since abnormal
expression
of19 DEJ-1Acknowledgments
WethankA.Pisani forherexperttechnicalassistance, C. Minghelli's
for artwork, and N. Labate for typingthemanuscript.Wewishtothank
P.Marinkovich forcommentsandfruitful discussions.We aregrateful toDrs.L.Bruckner-Tuderman,D.Woodley, I. M.Leigh,D.Gerecke, R. Burgeson, and P.Marinkovichfor providing antibodies;Dr. R. Bur-gesonforthe KAL-55 cDNA probe; andtoPr. Y. Dumez,Dr.M.P.
Cordier, and Pr. Thoulon for providingsamplesofH-JEBfetal skin.
This workwassupported by the followinggrants:INSERM-Caisse Nationale d'Assurance Maladie des Travailleurs Salaries, Fondation pourlaRecherche Medicale (France); Dystrophic Epidermolysis Bul-losaResearch Association foundation; Association Franqaisecontre les Myopathies; Association contre les Epidermolyses Bulleuses Hereditaires.
References
1. Fine, J. D., E. A. Bauer, R. A. Briggaman, D. M. Carter, R. A. Eady, N. B. Esterly, K. A. Holbrook, S. Hurwitz, L. Johnson, A. Lin, R. Pearson, V. P. Sybert. 1991.Revised clinical and laboratory criteriaforsubtypesofinherited epidermo-lysis bullosa. A concensus report by the subcommittee on diagnosis and classifica-tion ofthe national epidermolysis bullosa registry. J. Am. Acad. Dermatol. 24:119-135.
2. Lin, A. N., and D. M. Carter. 1992.Epidermolysisbullosa.Basic and clinical aspects SpringerPublishingCompany, New York. 295 pp.
3.Uitto, J., and A. M.Christiano. 1992. Moleculargenetics of the cutaneous basement membrane zone.Perspectivesonepidermolysisbullosa and other blis-tering skindiseases. J.Clin.Invest.90:687-692.
4. Fuchs, E., and P. A.Coulombe. 1992. Ofmice and men:geneticskin diseasesofkeratin.Cell. 69:899-902.
5. Epstein, E. H., Jr. 1992. Molecular genetics ofepidermolysis bullosa. Science(Wash. DC). 256:799-804.
6.Sakai, L. Y., D. R. Keene, N. P.Morris,and R. E.Burgeson.1986. Type VII collagenisamajorcomponentof anchoringfibrils. J.CellBiol. 1577-1586.
7.Uitto, J., L. C. Chung-Honet, and A. M. Christiano. 1992. Molecular biol-ogy and patholbiol-ogyoftype VIIcollagen.Exp. Dermatol. 1:2-11.
8. Ryynanen, M., R.G. Knowlton,M.G. Parente,L.C.Chung,and J.Uitto. 1991. Human type VII collagen: genetic linkageofthe gene(COL7A1) on chro-mosome 3 to dominantdystrophicepidermolysisbullosa. Am. J. Hum. Genet. 49:797-803.
9.Hovnanian, A., C. L. P. Duquesnoy, C. Blanchet-Bardon, R. G. Knowlton, S.Ansellem, M.Lathrop, L.Dubertret,J.Uitto,and M.Gossens. 1992. Genetic linkage of recessivedystrophic epidermolysisbullous to the type VIIcollagen gene.J.Clin. Invest.90:1038-1046.
10.Christiano, A. M., D. S. Greenspan, G. G.Hoffman,Y.ZhangTamai, A. N. Lin, H. C. Dietz, A. Hovnanian, and J. Uitto. A missense mutation in type VII collagen in two affected siblings with recessive dystrophic epidermolysis bul-losa. Nature Genetics. 4:62-66.
11.Tidman, M. J., and R. A. J. Eady. 1986. Hemidesmosome heterogeneity injunctional epidermolysis bullosa revealed by morphometric analysis. J. Invest. Dermatol. 86:51-56.
12.Garrod, D. R. 1993. Desmosomes and hemidesmosomes. Curr. Opin. Cell
Biot.5:30-40.
13.Schwartz, M. A., K. Owaribe, J. Kartenbeck, and W. Franke. 1990. Des-mosomes and hemidesDes-mosomes: constitutive molecular components. Annu. Rev. Cell Biol.6:461-491.
14.Riddelle, K. S., S. B. Hopkinson, and J. C. R. Jones. 1992. Hemidesmo-somesin the epithelial cell line 804G: their fate during wound closure, mitosis and druginduced reorganization of the cytoskeleton.J. CellScience. 103:475-490.
15. Li, K., G. L. Giudice, K. Tamai, H. C. Do, D. Sawamura, L. A. Diaz, and J.Uitto.1992.Cloning of partialcDNAsformouse180 kDa bullouspemphigoid protein of the cutaneous basement membrane zone. J. Invest. Dermatol. 99:264-270.
16. Ryynanen, M., R. G. Knowlton, M. Kero, D. Sawamura, K. Li, G. J. Guidice, L. A. Diaz, and J. Uitto. 1991. Bullouspemphigoidantigens(BPAGs): identification of RFLPs in humanBPAG1andBPAG2, and exclusion as candi-date genes in a large kindred with dominantepidermolysisbullosasimplex. Geno-mics. 11:1025-1029.
17.Kurpakus, M. A., and J. C. R. Jones. 1991.Anovelhemidesmosomal plaque component, tissue distribution and incorporation assembling hemides-mosomes in an in vitro model. Exp. Cell Res. 194:139-146.
18.Hieda, Y., Y. Nishizawa, J. Uematsu, andK.Owaribe. 1992. K.
Identifi-cationof a newhemidesmosomal protein,HD1:amajor, highmolecularmass component of isolated hemidesmosomes. J. CellBiot. 116:1497-1506.
19.Stepp, M. A., S. Spurr-Michaud, A. Tisdale, J. Elwell, and I. K. Gipson. 1990. a6,34 integrin heterodimer is a component ofhemidesmosomes. Proc. Nati. Acad. Sci. USA. 87:8970-8974.
20. Quaranta, V., and J. C. R. Jones. 1991. The internal affairs of an integrin. Trends Cell Biol. 1:2-4.
21.Sonnenberg, A., J. Calafat, H. Janssen, H. Damms, L. M. H. Van der Raaij-Helmer,R.Falcioni,S. J. Kennel, J. D. Aplin, J. Baker, M. Loizidou, D. Garrod. 1991. Integrin a6j34 complex is located in hemidesmosomes, suggesting
amajor role in epidermal cell-basement membrane adhesion. J. CellBiol. 113:907-917.
22. Jones, J. C. R., M. A. Kurpakus, H. M. Cooper, and V. A. Quaranta. 1991. Afunction for the integrina6j#4in the hemidesmosome. CellRegul.2:427-438. 23. Verrando, P., A. Pisani, and J. P. Ortonne. 1988. The new basement membraneantigen recognized by the monoclonal antibody GB3 is a large size glycoprotein:modulationof its expression byretinoicacid. Biochim. Biophys. Acta.942:45-56.
24. Verrando, P., B. L. Hsi, C. J. G. Yeh, A. Pisani., N. Serieys, and J. P. Ortonne. 1987. MonoclonalantibodyGB3, a new probeforthe studyof human basement membranes and hemidesmosomes. Exp.CellRes. 170:116-128.
25. Rousselle, P., G. P. Lunstrum, D. R. Keene, and R. E. Burgeson. 1991. Kalinin: anepithelium-specificbasement membrane adhesion molecule that is a component ofanchoring filaments. J. Cell Biol. 1 14:567-576.
26. Marinkovich, M. P., P. Verrando, D. R. Keene, G. Meneguzzi, G. P. Lunstrum, J. P.Ortonne, R. E. Burgeson. 1993. The basement membrane pro-teinskalinin and nicein are structurally and immunologically identical. Lab. Invest.69:295-299.
27.Marinkovich, M. P., G. P. Lunstrum, R. E. Burgeson. 1992. The anchor-ing filament protein kalinin is synthesized and secreted as a high molecular weight precursor. J.Biol. Chem. 267:17900-17906.
28.Vailly, J., P. Verrando, M. F.Champliaud,D.Gerecke,W. D.Wolf,C. Baudoin,D.Aberdam, R. E. Burgeson, E. A. Bauer, and J. P.Ortonne. 1992.The 100 kDa chain of nicein(BM600)/kalinin is a laminin B2 chain variant. Eur. J. Biochem. In press.
29.Gerecke,D., D. W. Wagman, A. M.McDonough,and R. E. Burgeson. 1992. cDNAsencodingfor the three chains ofanchoring filament proteinkalinin showsimilaritytothe lamininA,BI and B2 chains.Mol. Biol.Cell.3: la.(abstr. 2.)
30.Baudoin,C.,M. F.Galliano,P.Verrando,J. P.Ortonne,and G. Mene-guzzi. 1994. The identification of a clonecorrespondingtothe 150 kDa of nicein, revealshomologies withthechain Aoflaminin. J. Invest. Dermatol. In press.
31.Kallunki,P., K.Sainio,R.Eddy, M. Byers, T.Kallunki,H.Sariola,K. Beck,H.Hirvonen,T. B.Shows,and K.Tryggvason. 1992.Atruncatedlaminin chainhomologous to the B2 chain: structure,spatialexpression,and chromo-somalassignment.J.CellBiol. 119:679-693.
32.Marinkovich,M.P., G. P. Lunstrum, D. R. Keene, and R. E. Burgeson. 1992. Thedermal-epidermal junctionof human skincontainsanovellaminin variant. J.Biol.Chem.119:695-703.
33.Carter,W.G.,M.Ryan,and P. J. Gahr. 1991.Epiligrin,anewcell adhe-sionligandforintegrina3fl1 inepithelialbasement membranes.Cell. 65:599-610.
34.Fine,J.D.,Y.Horiguchi,J. J.Jester,and J. R.Couchman. 1989. Detec-tion andpartialcharacterization ofamidlamina lucida-hemidesmosome-asso-ciated antigen (19-DEJ-1) present within human skin. J. Invest. Dermatol. 92:825-830.
35. Wayner, E. A., S. G. Gil, G. F.Murphy,M.S.Wilke,and W.G. Carter. 1993.Epiligrin,a component ofepithelialbasementmembranes, isanadhesion ligandfora3Bl positive lymphocytes.J.CellBiol. 121:1141-1152.
36.Amagai,M.,G. W.Elgart,V.Klaus-Kovtum,and J. R.Stanley. 1991. Southernanalysisof the 230-kDa bullouspemphigoid antigengene in normal humans,animals,andpatientswithjunctional epidermolysisbullosa. J. Invest. Dermatol. 97:248-253.
37. Nazzaro,V.,E.Berti,A.Cerni,A.Brusasco,R.Cavalli,and R.Caputo. 1990.Expression ofintegrinsinjunctionalanddystrophicepidermolysisbullosa. J.Invest.Dermatol. 95:60-64.
38.Jonkman,M.F.,M.C. J. M. deJong,K.Heeres,andA.Sonnenberg.
1992.Expression ofintegrin a6,B4injunctionalepidermolysisbullosa. J. Invest. Dermatol. 99:489-496.
39. Heagerty,A.H.M.,A.R.Kennedy,R.A.J.Eady,B.L.Hsi,P.Verrando, C. J. G.Yeh,and J. P. Ortonne. 1986.GB3 monoclonalantibodyfordiagnosisof
junctionalepidermolysisbullosa.Lancet.8485:860.
40.Schofield,0.M.,J. D.Fine,P.Verrando,A.H. M.Heagerty,J. P. Or-tonne, and R.A.J.Eady.1990.GB3 monoclonalantibodyfor thediagnosisof junctionalepidermolysisbullosa: resultsofamulticenterstudy.J. Am.Acad. Dermatol. 23:1078-1083.
41.Verrando, P., C.Blanchet-Bardon,A.Pisani,L.Thomas, F.Cambazard,
R. A.J.Eady, 0.Schofield,and J. P.Ortonne. 1991. MonoclonalantibodyGB3 definesawidespreaddefect of several basement membranes andakeratinocyte
dysfunctioninpatientswith lethaljunctional epidermolysisbullosa.Lab. Invest. 64:85-92.
42. Meneguzzi, G., M. P.Marinkovich, D. Aberdam, A. Pisani, R. Burgeson, and J. P. Ortonne. 1992. Kalinin is abnormally expressed in epithelial basement membranesof Herlitz's junctionalepidermolysis bullosa patients. Exp. Derma-tol.1:221-229.
43. Verrando, P., and J. P. Ortonne. 1993. Antibodies to the basement mem-braneproteinnicein(BM-600)providenewclues in lethaljunctional epidermo-lysis bullosa. J. Invest.Dermatol. 101:738-743.
44.Fine, J. D. 1990. 19-DEJ-1, a monoclonal antibody to the hemidesmo-some-anchoringfilamentcomplex, isthe only reliable immunohistochemical probefor all major forms of junctional epidermolysis bullosa. Arch. Dermatol.
126:1187-1190.
45.Ortonne,J.P., P. Verrando, G. Yeh, B. Hsi, A. Pisani, and F. Bernerd. 1990. Theepidermal antigen recognized by monoclonal antibody GB36 is very lateantigen 6 (VLA-6). J. Invest. Dermatol. 94:400. (Abstr.)
46. Rheinwald,J.G., and H.Green. 1975. Serial cultivation of strains of humanepidermal keratinocytes:theformationof keratinizing colonies from sin-gle cells.Cell.6:331-344.
47.Simon,M.,and H. Green. 1985.Enzymatic cross-linking of involucrin andother proteins by keratinocytes particulatesinvitro. Cell. 40:677-683.
48. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head ofbacteriophageT4. Nature(Lond.).277:680-685.
49.Schomczyski,P., and N. Sacchi. 1987.Single-stepmethodof RNA isola-tionby acidguanidiumthiocyanate-phenol-chloroformextraction. Anal. Bio-chem. 162:156-159.
50.McIntosh, I., A. Hamosh, and H. C. Dietz. 1993. Nonsense mutations and diminishedmRNAlevels.NatureGenetics.4:219.
51. Peltz, S. W., and A. Jacobson. 1992. mRNAstability:intrans-it.Curr. Opin.CellBiol. 4:979-983.
52. Hunter,I., T. Schulthess, M. Bruch, K. Beck, and J. Engel. 1990. Evidence for aspecificmechanismof laminin assembly. Eur. J. Biochem. 88:205-211.
