Transient Expression of Epidermal Filaggrin in Cultured Cells Causes Collapse of Intermediate Filament Networks with Alteration of Cell Shape and Nuclear Integrity

Beverly A. Dalc, * t1:~1 I't.ichard B. Presland, *§1 S. Patrick Lcwis, * Robert A. Underwood.~ and Philip FleckJ11an~ Departments o f ' 0 .. ,,1 Diology. j- Pcriodollti c'. :j:Diochcmislry. and §MedicineiDenm\l o lo)(y . Ull ivcrsiry of Wash illg to ll . Scanit-.

Washington. U.S.A.

Filaggrin is an intermcdiate filal'l1ent-associatcd pro­tein (IFAF) that aggregates epidermal keratin fila­ments ;/1 ,,;tl'O and is thought to perform a similar function during terl'l1inal differentiation ;/1 "i"o. To test this function in living cells, we transicntly ex­pressed constructs encoding human filaggrin in both simple epithelial cells (COS-7) and rat keratinocytes. Scanning laser confocal microscopy showed that filaggrin-positive cells had collapsed keratin and vi­mentin intermediate filament (IF) networks, and that filaggrin partially co-localized with thc IF networks. Filaggrin was also dctectcd diffusely in the cytoplasm and nucleus. In contrast, when profilaggrin-like con­structs, containing five filaggrin domains separated by the linker scquences, were expressed in cultured cells, immunoreactive granules fonned. This finding is reminiscent of the insoluble nature of nativc pro-

I Iltenlwdiate filamelll associated protei ns (IFAP) arc a di ­verse gro up of structural proteins t1wt playa role in the l11 ac ro mol ecu l:,.r orga nization and funct"ion o f intermediate filaments (11"). an impo rl·ant compo ncnt of the cytoskelc·ton (reviewed by Albers alld IOuclts. 1992). An IFAP ma y

co-distdbutc with I Fs ill ce ll s o r be prese nt at I F anchorage sites; it may co-puriry with IFs ;lIld affect filam ell( org:lIIi za ti on . il sselllbly and/or ma cromol ec ular stru cture (for rev iews, sec Fo isner and Wiehe. 199 1; Yall g 1'1 111. 1993: Presland . 1996). fi laggrin . a cationic protein Of l1l3f11l11 aJiall epidermi . WilS onl' of th e hr5t IFAPs to be chara ctcl'ized biochellli ca lly and tlill cti onall ),. It "~grl'gates with kenltin filaments, as we ll as other types of IFs . leading to dll'

formation of hi ghl y o rgani zed hUl1dl es or macrofib ril s: it was subsequently nam ed filaggrin for thi s ji/llll1ent n.t!..~rcgat;"g activit)'

Man uscript [l'c(' ivco .July 29. 19<)6: rev ised September 17. 199(,; accepted for p ublication Octoher I I . 19%.

Repri nt rcqu<' sts «) : 1)1' . D.A. Dale. l)e l' artl11 ent of Oral Biolog)'. University ofWa., hingto ll. Box 357 132 . Scatll e. WA 9H I9S-71.'2 .

T his wo rk lVas prl"enlcd in part al the Society of In vestigative DC1'IIln ­

tology m e",.i,, !!;s ill 1995 alld 19\}(1. A bbreviations: IF. illtcnllcdi ," C ,·ilam cllt ; IFAP. illl crm ccli"le ti lall ll'l1l­

assoc ia ted prntcin ; IlEKs. ,.,11 ""idl' 1'II1IlI kenlli " cyte, ; I)AP!. ,, ' .6- dia­midi no-2-phcllylilldolc dihycl mchloridc .

I The first anel secol1d "ull lOr< cOl1lrihu tl·eI elJually In thi s Wl1I'k .

filaggrin that accumulates in keratohyalin granules ill IIi"o, suggesting that the linker peptides (present in profilaggrin but not filaggrin) are important for gran­ule formation. Cells expressing filaggrin also dis­played disruption of thc nucleus and the nuclear envelope; they rounded up and lost attachment to the substratum, in contrast to control cells ove.r-express­iug (:J-galactosidase. This functional test of filaggrin in living cells supports its role in the reorganization and packing of keratin IF in epidermal differentia­tion. Moreover, the observed effects on cellmorphol­ogy and nuclear integrity suggest that filaggrin may contribute to the fonn of apoptosis associated with terminal differentiation in epiderntis. KI')' II101'ds: illter­mediate .filamellt -assIJCiated proteill/ epidemlis/al,"pto.<;s.

] !tlllest D el"l"atol 108:179-187, 1997

(Dale {'( nl. 197!l; Ste inert cl nl. 1 <)8 I) , Filaggdn W<1S proposed to tilll ctioll as till' interfibmcntom m atrix protein of the stratulll corneull1. based on its abi li ty to aggregate keratin Irs ill .. ilm into lI1acrofibrils that till the anuclcated cells of the stratulI1 cornelllll (Da le 1'1111. 1(78).

Filaggrin is syn thes izt.'d in the granular ce ll layer of epi,krmi, as

a large. inso luble. and highly ph osphory lated pn'cllrsor. protlla!!­grin , which is loca lized in kcra roh),il lin granules and forms the bulk of thei r ('(lIItent (Steven /'1 nl. 199(); Manabe rI nl. 1991) . Profilag­gri n consists of l1Iul ti ple tila ggrin rept'at' tlanked by uniquc - and

- tcnll inal dOl1laills (Haydock 'Illd Dale. I <JH(i: Ga n ct til . 19<)() ;

Pres land ('I nl. 19(2) . ach f'ila gg"i n repeat consists of a carionic st.'quence with a short hydrophobic linker peptide . During renninal differentiation. protilaggrin is Jephosph oryla ted and proteolyticall) processed. resul ting ill the re moval of the,e h ydrophobic linker peptides, as well a the flanking N- and C-terminal domains. ro generale marun' fila ggrin (Resing 1'1 nl. 1989; 1993a: 199311: reviewed ill Dale 1'1 nl. 1994).

Evidence supporting the fun ction of Iilaggrin ;11 ";1'<' includes the obse, vatio ns that flla ggrin (but not proti laggrin) is capahle or a!!gregating kerati n lFs (Lonsdalc- celcs /'t III. 1982; Harding and

cott. 1983) : Ihat tilag!!rin is expresst.'d only in kcratinizin!! epithe­lia ( ' mith and Dall- . 1986), and that prcmaturl' expression of filaggrin is dfective ly prc \'e l1tcd in livill ~ cell layers oftlte epidermis

0022-2 Il ZX! 97 I S I 0 .50 • Copyri gh t \~ 1997 by 'hl' Sncil'ty for II1 v<·.,ig3ti,·,· I)cnll .lI n l"ln. II1 c.

179

180 DALE £1' AI.

by expression as an inactive prec ursor that subsequently undergoes extensive processing (reviewed in Dale e/ ai, 1994) . Immlilloelec­trOll microscopy of epidennis also suggests that filaggrin is an IF-bundling protein ill Ilivo. Hence, the tight p ,lcking of keratin IFs into mac rofibril s coincides with the di ssolution of keratohya lin granules, which occurs with the processiJ1g of profilaggrin to form filaggrin. The association of filaggrin with keratins may faci litate disulfide bond f0n11ation betweell keratin polypeptide chains and e nable them to survive the massive remodeling that e n sues with tenninal differentiation . Subsequent chemical modifica tion and degradation of filaggrin. i.e ., the loss of filaggrin immunoreac tivity in the upper stratum corneum, coincides with the "loosening" of k e ratin IFs (M3nabe er ai, 1991) and the degradation of filaggrin to free amino acids , which are thought to bind water in the uppe r stratum corneum (Rawlings er al. 1994).

The association of filaggrin with keratin IFs to form macrofibrils has b een d emonstrated ;11 v;rro using purified filaggrin (Dale e/ ai, 1978; Steinert e/ ai, 1981) and filaggrin-like peptides (Mack e/ ai, 1993) . Nevertheless, the role of the protein in filament aggregation in tenninal differentiation of epidermal cells ;11 /1;/10 is still contro­versia l. This is based on its poor expression in some pathologic conditions (Manabe e/ a/. 1991) and on its possible alternate or additional fi.ll1 c tions (Rawlings el aI, 1994; Steinert and Marekov, 1995). The reorganization of th e keratin IF n e twork in the transition from the granular to cornifie d cell layers could result

from multiple factors, but the timing of profilaggrin processing makes filaggrin a strong candidate to fUll c tion as an IFAP ;11 ,,;/10.

Thus. it was important to demonstrate that filaggrin function s in living cells and is capable ofleading to a lte ration of the cytoskeleton in cell s in which it is expressed.

In the current study. we sought to d e termine whether filaggrin functions as an IFAP using transient transfection as a means of

ectopic prote in expression in two types of cultured epith e li a l ce ll s and by examining its effect on the organization of the IF cytoske l­

eton . We ha ve a lso investiga ted the ro le of th e linker peptide in modifYing the action of filaggrin in ce ll s b y u sing a profila ggrin-like form of the prote in . W e report here that expression of human filaggrin leads to the collapse of both keratin and vimentin IF networks in COS-7 cell s and of keratin Irs in epiderma l keratino­cytes. Further, we show that cell s expressing filaggrin have a di srupted nuclear envelope . We also demonstrate that expression of proftlaggrin-like constructs that include linker peptides yie ld an apparently less solubl e protein, which fonn s granu les within the cell s.

MATERIALS AND METHODS

Preparation ofFilaggrilt Constructs Human filaggrin constructs were pre pared both by a polymerase chain reaction (PCR}-based approach and by the direcr cloning of human filaggrin repeats into an expression vector containing an '-'-terminal FLAG sequence. which included an ATG initia­tion codon (Fig 1). Expression vccto", containing cither the cytomega lo­v irus (CMV) immediate early promoter (pcDNA3. In vitrogen. San Diego, CAl or the involucrin promoter (H3700-pL2) , a generous gift of Dr. J. C .. rroll (Imperial Cancer R csearch Fund, London , U . K.). were used . The involucrin prol11oter pl asl11 id drives high levels of /3-galactosidase exprcs­lion in keratinocytes (Carroll ,md Taichl11an, 1992: C arroll el ai, J 993), whereas the C M V prol11oter gave high levels of expression in COS-7 ce lls and va riabl e expression in a rat cpidcrnlal keratinocyte ce ll li.ne (REKs).

C OlIs(mcrioll ,,( I'C-L .",1 FG+ L Two PC R-derived cOl1<tructs wcre pre­pared cither with (FG + L) or without (FG-L) the linker sequence that flanks each fi laggril1 domai n in profilaggrin (Fig 1). For tbjs study, the linker sequence was defined as the peptide RSGRSGSFLY . which correspo nds closely with the boundaries of the linker rccentl y defined by mass spec­trOl11ctry (Thulin and Walsh. 1995) . PCR was performed using the oligonucleotides 5' -CCCGCGGCCGCACCA TCCAGGTGAGCACTCA­TGAACAGTCTGAG-3' ( - linker) and S'-CCCGCGGCCGCACCAT­GAGGTCTGGACGTTCAGGGTCTTTCCTC-3' ( + linker): the coml11on downstream oligonucleotide consisted of S'-CCCGCGGCCGCTTATC­CCCCTGA CCGGTCACGTGCGGACTC-3' . The o ligonucleotides in­clude NIlII re triction sit ~ for cloning (underlined) and artificial translation initiation and termination codons (italicized). PC R was performed for 30 cycles of I min :,t 94 °C, I min at 63°C , and 1 min at 72°C in a Ampli tron

T ilE JOURNAl 01' INVI.STIGi\TIVE D ER.MATOLOGy

ATG TM

~ FG - L ~

ATG TM

~ FG+L ~

ATG TM

FLAG-FGl _ FLAG

ATG TM

Number Of AminoAc~

314

324

324

~~ f;I I f;I I f;I I f;I I e II hGH polyA FLAG-FG5 ~ . - . 1620 FLAG

Figure L Humalt filaggrin expression con struets, 0 indicates Se,

quence of m:ltllre fonn of tilaggrin: m indicate\ thc linker ,equence; C] indicates the FLAG cpitope. The FG- L and FG I L constructs were als prepared utili zin g the involucrin pro.I1l(Her (or ,tudie, in R.EK,. FLAG-FG~ and FLAG- FGS were detectable w 'th <l nnboche, to human fila g!;TI n and antibodies to the FLAG epitope. A/lalogou< COII< trll cts we re prepared witl) fi taggrin in the a l1ti-~c I1 SC orientation. NlIIllbcr of ,Ill1ll10 acids indi atcd docs nol include the FLAG cpilOpe (eight r(',idlle,) .

I thcrmocyc1er (Barn,tcad-Thcnno l),lI e, Dubuque. IA) lI ~ ill g the profilag, grin genomic cione com dd 211 . 1 as template ( Pre~I;, "d cr nl, 1992). The PCR product (- 1 kb) WaS gel purified , digested w ilh Noll . alld cloned intI) pcDNA.I, o r into H3700-pL2 afLer rCllloval of II", /3-g:l lactns idase gene with Noll . The FG-L con,truct ellcodcs the mature tilaggrin protein (3 14 amino acids), w here", the FG + L con'truct encodes a \ing1c filaggrin (324 amino acids) dOll1ain wit h a linker at Ihe N-tenninll\, ,i .llil:lr to w hot would b~ expected during ;/1 IIit)I' procc" ill ~ .

COlls/mrlioll 4 FLAG-FG J nllt! I·LAG-Fe 5 rill' pc DNAJ -FLAG ma111, l11 ali an expression vector was prepared by doning th l' multiple clo ning sire and surroundin g lequcncc\ ofpFI AG-2 (1131 i<odak, Ncw H aven , CT) into peONA3. Thi, was acco ll1pl ished by PC R u<ing pril11ers flank ed by KplIl and Apal sites lO faci li tate direct cloning of the I'I-LAG-2 sequence into pcD NA3. The leq ucnce o f die re\ulting pel )NA:I-FLAG po lylinkcr wa, verified by DNA seq uencing: the hylwid "ector contain ed :In N-tcnninal FLAG sequence including an initiation codon . Ihe 1I1uiliplc cloning site &On, pFLAG-2, and downstream stop codons in all three rc"ding frallle'.

The o ne- and fivc-filaggrin domain con,trllct, were prepared as follows. Two different plasl11ids (pH X I and pHX5) contai ning sing lc hUl11an fi lag_ grin do main< frOIll cosmid 28. 1 wcre digested wi th X/w i and doned into pcON A3-FLAG. Si ll1ilarly, cO'll1id 28. 1, which contains the 3' - half of the profi la g~,'rill coding sequence (Prcslallli 1'1 til, 1992). wa' digcsted with EwRI, cnd filled with tilt' I<l cnow fragmellt of DNA polymerase I, and cloned illto the EcvRV site of pcD NAJ-FLA(; . T he resultin g COllStrUct, pFLAG-FGS, contailled five' t,laggri ll domai ,,,. PI,,\lllid DNA was prepared by alka line lysis and purified usi ng Qiagen SOO co lull1ns a' directed by the l11anufacturer (QIAGEN, C hat,worth, CAl. All cOll structs were ve rified by sequencing. Thcse construct, ill w hich the linker '''q ucll ec is internal encode proteins si lllil ar to profi laggri ll ill "i'/II.

The express ion constru ct for /3-ga lactosid:l.<e W ;I< pSV-/3gal (Prolll cga, Madison, WI) .

Cell Culture and Transfection Procedures COS-7 African Green monkey kidne y ce ll , were cul tured in Dulbecco's llIod ifi ed Eagle 's lI1edium supplemented with 10')1" fctal boville 'eru l1l alld werc passaged weekly. RE Ks. a rat epidermal keratillocy .. e ce1l lill e (n gene rOil s gi ft fi·o lll Dr. Howard Baden . Massachu,eLt< Gcneral Hm pital, lIoston, MA) were grow" as previously described (Baden and Kt/bilu,. I <JH.3: Il aydock 1'1 nl, 1993). All ce ll Ijnes were tested to exci llde the poss ibility of 1I / ),( <'I'/"SlII" contamjn3-cion.

COS-7 cells and IlEKs were tra",fe'ckd ill (,11-111111 dishe, at 60% confluence using LipofcctAM INE reagc lll (Life Tl'chll () logic" Gaithers­burg. M 0) essenti all y as recomlll ended by the Ilwllt/fae-turer. For c;lch

VOL. 10B. NO. 2 FI?HRU I\R Y I 'I'l?

[ransfection , 2.5 ILg of fi laggrin DNA and 2 ILg of pSV -/3g"1 wcr" mix ed with 40 !J.g lipid I'cagen t ano serum-free Dulbccco ' < modified E"gle's medi un, pCI' 1111 alld incubated Ht 37°C lor 'I h . Alter 'I h . all equ<" vo lul11e of20% Dulbecco's 1I10dified Eagle's Illed iull1 was added . the suspl'nsioll was incubated ovcrniglH ~t .3 7°C. ;111d the l1\e diurn was ch~n~clt. Approxiln:l te\y -18 b aflcr lrnllslccrioll. thc cell, wcre harvested alld washed ill co ld phosp h a tc-bulfc red sa line. and Iysates were prepared ill L1.25 M Tris(hy­droxYll1e th yl) nll1illo l1l cthnne/ II C I. pH 7.8. alld assayed It)r ,a-ga lactosidase activ ity as dcscriln'd ('a ltlbrook ('( ,rI. 1989). For illlllllllloAuorcscencc. approximately 1-4 X 10 1 ce lls were plated 0 11 glass coverslips ill 12-wcll plales and trnll sfccted as above . Cells were wash co ill pl ,o'l'hntc- blllrcrcd sa line . fixed. alld pcrllleabili7ed ill rnetl","ol :acetollc (:l: I ) for 10 min at - 20°C, rinsed ag:lill ill phosphatc-bullc red s;l lille , alld ston'd nt 4°C lIlltil antibody labclillg was performed. Filaggrin sense and allti-'c",e constructs were used ill par"lId in "II tr"nsfectioll experi lll ellls cOllductl·d.

Antibodies T he 111 01lnclOll:11 (AKHI . Dale "1 ,,'. 1987) "lid I'o lyclo ll al antibodic< (8959. Flcck l""" f'f til. 1985) 10 11I1I1Iail ,·ila!(/!'rin " li d the polyclon a l aillibody to mt profilagwin/ fila ggrill (466. H:lydock alld Dale. 1986; Haydock ,.( " I. 1993) were developed by our laboratory. T he monoclo llal antibodies AE I ,"Id AE.3. which arc spl'cifir for ac id ic and basic subfanu li es of keratins. respeclively, wcre a gcnel'Ou, gift of J)r. T .-T. SUIl , NYU M e dic;11 Center. N ew York (Eichner /,( <II. 1(84). O ther monoclonal and polyclonal " "lihodies were obtallled commercia ll y and included mono­clonal a"tibod ies to keratin 8 (used for COS-7 cell srud ies). vimen ti n. alld f3-rubulin (Sigma. St. Louis. MO). lami n 13 (Novocastra. Newcasde- upon­Tyne . U . K.). " lid the :lllti-FLAG :1lltihodics M2 and M 5 (1131 Kodak) . Polyclona l antibodies inclllded panke'ratill (13iuGcllex. San R ;IInoli. A) and anti-i3-ga lacro.<idase antibody (US Uiochcmicals. Cleveland . II) .

Gel E lectrophoresis and Inullunobiot analysis C ultured ce ll s trallS­rceted "'ith fi laggri ll CO llstructs wcre ex tracted with 0.25 M Tris(hydroxym­ethyl)amiI1Oll1ctlwne / H C I. I'l l 7.8. ill rhe ah"'ll cl' of detergent or tlre:l, alld proteins werc <eparated 0 11 7 .5'Y.,- 12'Y., Sn S-PAGE gels; Tris(hydroxYlllcth­yl)am ino l11 eth:mel llrc" extracts of hUIl,,,n forcski ll epidermis tlwc cOlltai ll profilaggrill and filaggrin were useo a< control< (Lacnnilli. I 'no; Sybert ,.( al. 1985). Proteins were hlo(('cd to nitroccl ildosc and ilnnllllloreactive proreins visualized as pl'ev ionsl y descrihed w ith th,' avidi n- biotin Itlethod using 4-chloro-I-r",phthol (Towhill t'f " , . 1979; Ilaydock ,'1 al . 1993) or cllhnl'l ccd chemilum inesccnce (ECL. AI II l'rsha lH. Arlinglo ll Heights. IL). ECL blots were developed on ECL II )'perlilnl (i\rncrshalll) .

[mm unotluorescel1ce Detec tion of Expressed Proteins and Other CeUular Constituents For double illlltllllloAnorescence. the first pri­mary antibody (InOlloclOlwl) WlIS inc lihatcd for 1 hand cl etc'ct"d with a secondary biotin- labeled horse anti- mo llse IgG (Vector Laboratories. Burlingame. CA; 1:4(0), :rnd <trcp tll vidill Texas Ren (V<,·c tor. I :BOO) wit h multiple rinses b .. rwe!!n iliClIbations . T he second prim ary (polycloml l) "'a' incubated for I h. thell rill sed '"ld oetected with tluo roscein i.sothi(lcy,nl"te (FITC)-Iabe led goat :I n ti-ra bb it Ig (Vector. 1:2(0) . S'nnples were co"er­slipped us;ng Vcctasbield 1ll001lltilig medium to minimize fadi ng . Image acquis ition wa< O il a 13io-Rad (RichnlOnd. CAl MIlC-(,OO 1:I,er scanning confoca l n'l.icroscopc " quipped with a krypton / argon- mixed gas laser lisen in dual excitation Ill ude passing ·IH8 nm ","ve len~th for FtTC excitation alld 568 nm "':lvc!c ngth for Texas R eo l'xcitalioll . Z-s tep thickness WllS n.s or J micron for 60 X : optica l section thjckl1css \va~ set ro giv~ SOl1le overlap between optical slice~. Bleed thro ugh fi'o l1l the red . hnllllt'l illro the ~recn chamlel "'as never observed. bUl hleed through from ~" CCIl into red w", observed occas ionall y alld sublracted using the Uio-Rad COMOS 1)- l3lcl·<1 program. fmage tiles we re split alld taken into Adobe Photosh,,!, \'0 djsplay image in nlol' as side b y side or Illl'l"gcd ~di n's or Ill il X iuHl1l1 prqjcctions. Color prints wen' olllputted to a Tektrollix dy,' subl il11atio ll printer nt 150 dpi. POI' D NA st.illing. sa mples previously sl.nill ed lor filag/!,rin were incub:tted ill 0.00 I 'XI -I' .6-diamidino-2-phcnylindolt- dihydrochloridc (DAPf) for 10 Illill lhc n w:" lu.:d with phosph:ltc-bufic rcd sa line. DA PI stai ll was observed using a ultrav io let fi lter hy cpiflllorc<CClrcc 0 11 an O lympus BH-2 microscope.

RBSULTS

Single and Multiple-Domain Filaggrin Polypeptides Can Be Expressed in COS-7 Cells and Epidermal Keratinocytcs Tbe filaggl'in express io n cun structs used in thi s study arc shown in

Fig 1 . The fG+L and FG- L con structs prodtl Cl'd immunoreactive tilaggrin protein when expressed tran sien tl y in e ith e r COS-7 n~ ll s or REKs; the ex pressed prote in was s imilar in size to ,, "the lltie human filaggri n (Fig 2A). T he FLAG-FG COllstructS cOlltai nillg

either nc- or five-fi laggrill domains also produced prote in s of lit e

FG

REKs cos Figure 2. Ectopic expression of lilaggriu in cultured COS-7 cells 3nd REKs ana lyzed by immulloblot 3I1alys i., (A) Transfucion \\ ilb FC-L. FC + L, ann FC;(a~) showing expression ofth" .'l7-kDa prolein ill horh cell types. hu t nOI in the anti- ,ct"c control (as) . Il11l1\uuohlm detect.ion wa, h)' .Ividin-hiotin pcnn.ida~c cO l11plC'x . Norc rhat lh~ siz(' oCthe linb.cr j, (00

shorl to " It er migr:,tiol1 . and that the exp"·,,ion o( FC + L ill REI<. was WNk

and variabk. (/:!) :lUli (C) Transfl'crion with FLAG-FG I . LAG-I' GS. 'lIld FLAG-FC("<) in REK < (B) aud OS-7 ce lls (e) showing expression of olll'-ti l'lggrin (38 klh) and fiv<'-~I\agf!rin dOl11aill constnlCfS (190 k))a . doub let)(-). Control forl',kin cxtmCl <how< native I'rofilaggrin (1'\ and ti laggrin (FG) . Irnn ll itlodetl'ction using polyclonal :ltlt.ihod)' to hUIl1d1l fibggrin :Uld ECL; identica l results were ohtainl'n with "ntihodr M~ <I'",.ili,. for the FLAG epil.opc (not shown) . The FlAG-FG I protein llIigrat ... , ilion' . Iowly th"n l1ati"e fi laggrin due to the .lclditiotl of the cpiropc. Othcr b.lnd, (a \so oh~crvcd in lh~ .\t\ti -S{' USl' controls) ar~ nOl\!\I'cl'ific .

predicted molecular mass (38 kl)'l and - 190 kDil. respcctively. F ig

2B,C) . 1 roteolytic processing of the five-domain protein \Va< not evide n t by i,lllllut1obiot a n a lysis tinder the growth l' ondition< ttSen

(subconRue nt cultures were used to optimizl' il11nlllt1oRuor('~cence o bservatio n s) . Processing was anticipated ill the REK line but wn.

Ilot observed here. flerhaps bec~use these ceIls were not sufficiently dilferelltiated to express the n:quired enzymes or becau<e of species-specific asp,·cts of processing (Resing ('/ "I. 1989; 199.h).

N o fi laggrin expression was d e tected using lilaggrin anti-sen'l' constructs (Fig 2). lluman ti laggrin was not detCl·table in untl'an,­

fected COS-7 ce Il s or in R.EKs. Although R.EKs express rnt protil"ggrin ( - SOO kDa) and fi laggrin (42 kDa) alter conRuen ee

(H"ydoe k 1" "I, 1993). the rat protein docs not eros~-react with the hum an fil aggrin a n tibody used to llll'aSUrl' expression in transfccted ce ll s (dara (l ot shown). TIl<' cxpl'ession ofl.il;lggrin was delectable' ,1l

a ll t illl es between 12 and 72 h hut was maximal at 48 h ill 0 ,-7 cel ls and 72 h in lliKs (data not shown) .

Expression of Filaggrin in COS-7 Cells Resul ts ill Collapse of IF Networks Ana lysis of cells trallS(ceted with rhe FG- I and

FG I L t'ollStructs h y sca nning laser con Inca I microscopy rcycall'd a

182 DALE ET AL

striking reorganization of both vimentin (Fig 3A-q and keratin (Fig 3D-I) IF networks. often into perinuclear bundles or coils in contrast to the dispersed J F network in control ce lls (Fig 3J-L). In all filaggrin-positivc cells exam ined, the IF distribution was altered; this was never observed in control cells. The filaggrin staining was diffuse throughout the cytoplasm and nucleus. bue staining co­localized at least partially with vimentin or keratin IFs (e.g .• yellow/orange double immunofluorescence as in Fig. 3C,F,I). In COS-7 cells, the proteins encoded by both filaggrin constructs appeared to collapse the IF networks (compare Fig 3B and 3H to normal vimentin and keratin networks shown in anti-sense con­trols, Fig. 3K,L). These results suggest that filaggrin can function as an IFAP (or IF-bundling protein) in living cells. On the other hand, expression of filaggrin in COS-7 cells and REKs did not coll apse microtubules into perinuclear coils (datll not shown) , suggesting that the major effect of filaggrin was specifi c for the IF cytoskeleton.

The Linker Peptide Alters Filaggrin Function and Distribu­tion in COS-7 Cells and REKs The linker sequence is present between each filaggrin domain in profi laggrin and is removed by proteolysis during processing and formation of mature filaggrin (reviewed in Dale ef al. 1994). Two different types of constructs were used to test the effect of the linker peptide sequence on IF association and ce ll fi.mcrion; (i) a filaggtin domain with a linker sequence at the N-tenninus (FG+ L) and (ii) FLAG-tagged con­~tructs with the linker seq uen ce in an internal location with either one (FLAG-FG I) or five complete tilaggrin domains (FLAG-FG5) (see Fig 1) . The latter two constructs are more like profilaggrin ill

sitll, whereas the FG + L construct. with the linker at the N­tenninus. wou ld be present only briefly during processing of profilaggrin to ftlaggrin in transitional epidermal cells.

Expression of profilaggrin-like constructs that contain one or more linker peptide sequences resulted in fonnatio!] of protein aggregates of either rou nd or irregular shape in REK cells (Fig 4B,D) in contrast to the FG-L construct that lacked linker pep­tide(s) and was diffusely localized (Fig 4A) . Similar gramllar aggregates were ob erved in COS ce lls (Fig 4F, I) . In both cell types the immunoreactive gran ul es va ried in size and were present in the cytoplasnl and nucleus. They arc reminiscent of the immu­noreactive (profilaggrin-containing) gran ules in differentiating REKs (Fig 4q (also Haydock r l al. 1993). The gran ules detected in transfected cells , however. also reacted with a monoclonal antibody specific for the FLAG-epitope (Fig 4K), verifYing their ectopic origin.

Two types of t!vidence indicate that the expression of immuno­reactive granu les was specific for the filaggrin linker sequence ,1I1d not the FLAG peptide. First. the FLAG-FG(as) construct. which encodes a protein of 26 amino acids in length (including the FLAG peptide). never yie lded a granular staining pattern . Indeed. many investigators have used this hydrophili c epitope with no reports of a granular immllnofluore cence pattern of FLAG-tagged protein (sec for example, Kouklis el ai, 1994). Second, expression of the FG+ L construct, which lacks the epitope tag. also yie lded a g ranular imm unofluorescence pactern in REKs (Fig 4B). Con­structs with the linker iJlternal to filaggrin sequences (the structure most similar to native profilaggrin) always yie lded the granu lar pattern of staining. Intranuclear localization of filaggrin (but not keratins) was observed in varying degrees in these cells and other ce lls expressing all of the constructs tested (see section below).

Transfected cells expressing the profilagglin-like constructs also showed evidence of collap~e of I F networks. Keratin filaments were collapsed around the nucleus of positive R.EK cells (Fig 4E, an·olll). COS-7 cell s expressing FLAG-FG 1 display vario us degrees of IF disruption ranging ITom partial IF aggregation (Fig 4G,H arrow) to total disruption in hi ghly positive ce lls (note ce ll 3 in Fig 41,]).

Filaggrin Expression Results in Changes Associated with Cell Death As described above. ceLis expressing fuaggrin, par­ticu larly the mature fornl, showed aggregation and collapse of their fF networks. This finding was consistent with the predicted func-

TI l E JOURNAl Ufo INVrS IICA rlVIo DFltMATOLOGY

tion of filaggrin. A somewhat surpri~ing f-illding of these stl.ldie however. was that ce lls exprcs~ing tilaggrill di~rlayed changes il; shape and nll c lear integrity. In COS-7 cell s expressing tilaggrin, the cell cytop lasm was condcnsed; cell~ were (j'eqllcntIy rounded up and seemed to be lo~ing adherence to the sub~trntul11 (Fig SA,B). The change in cell shape was observed in cell~ expressing filaggriJI construct with and withollt the FLAG epitopc tag (compare Fig 4F,1 to Fig SA,B).

Cell s expressing ftlaggrin also showed evid ellce ofbl'eakdown of the nuclear co mpartmcnt. Both {3-galactosidase and filaggrin were detectable in the lIu cleus of filaggrin-positive ce lls (Fig SA,B,D; see also Fig. 3A, 4D); ill contrast. /:l-galactosidase was very rarely (if ever) observed in the nucleus ofCOS-7 ce ll ~ transfected with the {3-galactosidase pi;lsmid and/or the anti-sense filaggrin construct (Fig 5C, and not shown), demonstrating that nuclear disruption is not a nonspecific result of tramfection .

The presence of {3-ga lactos idase and fi laggrin in the nucleu s Of cell s expressing filaggrin uggested a disl"llption of the nuclear envelope in these ce lls. To further explore this phenomenon transfected COS-7 cells were stained with antibodies to humal~ filaggrin and nu clear lamin B (Fig 6) . III the most extreme cases. the nuclear envelope of ftlaggrin-positive cells was disassembled into star-shaped aggregates that reacted with the lamin antibody. This W,IS observed with both the FLAG-epitope-tagged construct\ (nlTvJIIs in Fig 6B) and the FG+ Land FG-L constructs (data not shown). In some ce ll s, lIuclei were condensed or irregular in shape (Fig 6F, arl'O III) , whereas in others filaggrill - reactive granules lined up a lon g the nuclear enve lope (Fig 6C,D). COS-7 cells transfected with an anti-sense constrllct (Fig 6G,lf) showed typica l nuclear envelope staining with some punctate st;l illillg within the nucleus.

Morphologi c evidence of nuclear condensation was a lso ob_ served in cell s expressing filaggrin constru ts . Transfected Cultures were stai ned for DNA with DAPI; filaggrin-cxpressing ceLis fre­quently had condensed and dysl1lOl'phic nllclei (Fig 7A ,B, arrows). Analysis of l11ultiple transfections showed that 49')1" (3 '15/648) of fi.laggrin-positive cells had condensed or abnormal nuclei compared co 11 °j" (637/5683) of ce lls tramfected with a control anti-sense construct (p < 0.0(1) (Fig 7Q. There W'IS no ~ign i fic'lI1t difference in tile percentage of conden sed nuclei in cells transfectcd with different tilaggrin sel1,e constructs.

01 CUS 'ION

Expression of Filaggrin Constructs in COS-7 Cells and EpidermaJ Keratinocytes Results in CoIl apse of IF Nct_ works Fila ggrin was th· first protein characterized for its ability to cause IF aggregation in an ill Ilil ro assay (Da le 1'1 al. 1978), and on that basis is now classed with a growing nllmber ofIFAPs (reviewed by PresJand. 1996). In this work we have tested the hypothesis that fi laggrin fi.mctions as an IFAP in living epithcli~t1 cells and demon_ strated that it has profound effects on IF distribution. suppo rting this h ypothesis. Expression of the mature form of ftlaggrin led to the collapse of the vimentin and kerati n IF networks into juxtanuclear aggregates in COS cells (Fig 3) ;l11d resulted in kerati ll filament coll apse around the Jlucleus in R.EKs (e.g .. Fig 4E) . The re. ults are consistent with the origiJlal filaggrin-kerati n mixing experiments conducted i ll Ii i!,.." which resulted ill formation of macrofibril-likc structures (I ale ('/ ai, 1978; Stei llert 1'1 ai, 1981). I" lIitro results, however, ca nnot a priori be extellded to the ill lI ill(l situation; therdare, our results :tre an importanf extension to demonstrate filaggrin function in Ji villg ce lls. The ill1mlllloAuorescenc~ tech­nique at the level of li ght microscopy does not prove direct associatioll of the proteins. and follow-up imnlllnoelectron micros­copy studies are needed to verifY the direct interaction . Neverthe­less, collapse of the IF network W'IS a commoll f'ature offt laggrin­positive ce ll s and was rarely observed in filaggrill-negative cells. In addition, Triton extraction of transfected cclls showed that a portion of th e expre~sed filaggrin remaincd in the detergellt­insoluble cytoske letal fi'nc tion , indicative of an IFAP (data not shown) . Taken together with the ill Il itro lIlixing experiments using purified proteins, the results strongly slIpport the interaction of

VOL. 108, NO. 2 H IIRLIAR Y I'J 'n III !I(:(:IUN c:,\PR.E~SION CALTS I·.S 11 COLI APSE 183

Figure 3, Filaggrin ('xpressioJl results in co ll apse or IF n(,tworks, Tr,II"fi''' ''''' C()~-7 eel" \\ ,'fl' ti",'d ~'l(l ,taillC'd h ' dnuhi<' irrlllll1llofiuorl'<('('ncc' with polycioll al alllihod)' to hUIl""' filag~rill (1'(;) :1lld '"0IloelOIl,,1 '"'Iihodi", to "'IIH'r "illll',,,ill (Vim) or k~ratill Il (K"r), ," illdkatl'd. Identical held, arc shown in (A) alld (Il), allli Ihe Iw,,- coIM ov,' rla} IC), ;r< , ., "'I a, ill (/)-1') all,\ I( :-1); (I) nlld I") ,In" the' \,11\\" fidd. ",hcr,' a' (/ ,) i, ,I ','paral,' ,' ;ew lA-C) "lid (D-I-"), PC-I. <,o",lnKI : (C- I), FG ·f I. <,,,mtrll,' t: (j- /.), 1-(;(;llIli- "'II"') c"",Im('1. Not(' ".,11,.1'''' orhlll h Villll'lIti ll "lid h'r,ltill IF lIt' twork, ill colltr"SI to control, (K (wd L) , Filagwill i, <,o- loc:li i7l'd with the' ('0 II " I''''d IF ("/1"'1/ ',<, IlIciJ l'.lIl'd hy nr,lI'gl' or )l'il(l\\' ('I,lnr d"Il('lIdillg Oil the' r('lan\'(' 'i!!nal illt"lISiry) ., well os

diffusely ill til(' <'),101'1""" alld Iludel" of PO,ill""' (',, \1<.

Figure 4. Expression ofprofilaggrin-Iike constructs yields granular immunoreactioll product in REKs and COS-7 epithelial cells. Cells were transfcctcd with constructs containing the linker sequence (B-K) or the mature filaggrin construct (A). fixed. ,,"d stained /,", humaJl filaggr.in (I'G) and keratim (Ker) . REKs: (A) FG-L: (B) FG + L; stained with polyclollal anti-human filaggrin; (C) sta ined wilh polycillnal anti- rat filaggri n Il) indicate endogenous profilaggrin in a stratified region of the cul ture: (D) and (E) FLAG-FG5. stained "'ing monoclonal anli-hu1l1an fil :.ggrin ami I'olycioll.d allli -pallkc ratin (the same fie ld is shown) . Note that immunoreactive granule. observed in ce ll s tram(ected wilh protilaggrin-like ("on'trucl, (H,D,r-,J) .lre ,illli lar lO endogenously expressed rat prufilaggrin (C). Also note partial co llapse or the REK keratin IF network (E. -). C S- 7 ce ll s: (F: /) lra"'(eeled with I' LAG-FG I: (K) , FLAG-FGS. (F:f) were stajned using polyclonal anti-filaggrin with an FITC-secondary and monodonal anti-kera lin 8 wi d. a Tex,\< 1 cd secondary: (H) is the two-color overlay of (F) and (C). (K) monoclonal antibody M2 to the I'LAG epitope (identical rendl . were ob,crvcd with MS. n()t , I.own). Note the partial collapse of the keratin IF network in (C,H): whereas in (1:f). three cells labeled 1, 2, and 3 ,how illl"rc~.sing "xpre«ioll or "LAG-FG I. wilh extensive di sruption of the keratin 1 F network in cell 3.

VOL. lOR . NO. I rBR UA I Y 1'1'17

Figure 5. Filaggrin expression results in alteration of eell shape and loss o[attachment to the substratum. Tramfcc(l'd COS-7 cell •. fixcd and stained for illllllullofluorc~cellt'l· \\ ilh poly!,!,)II,,1 alllihody to hUIll.II' ftlaggrin (rG), 01' polydollal ;ll'tibndy to I:l-galac(o~idasl' (J3-gal). (II), (IJ). alld (D) were tral1Sfectcd with the FC-L co",(ruc(, wlli:rl'as (e) W;l< i'ransiccIl'd with the anti-sense (FG(as)): all cu.lture< wcre cnrraml('ctcd wilh til<' ('xpreS<iOIl construct for i3-gaiactmidasl' . Exprl'«ioll "rthe ,'ctnpi" "rol"i" i< dctcctt'd ill the nucleu~ (,~,/:J,/)) "\ wl'll ii' the cywpla<Ill, \\ hcre<" 0111) C)'Wplil<lllic i3-galactosielase cxprc"io" i< detcl' (ed ill (( ;). Note (h ,'l till' til"ggI'ill-p'l'it;\,e ceUs in (A) and (B) havc cOlllpacl c)'LOI'I""" .1I1e1 "Itered ccll ,h.lpc .1I1d arc rni<,'d from the <uIfact'.

lilaggrin and IF •. T he tilaggrin- mediatecl alteratioll> did nor rc,ult in a genera l disruptioll of the cytmkeleton, as micrntubuks were relatively unaffected by tibggrin expre~,ion . Thl' dl'gree of IF aggregation or disruption may depend on the particular construct expressed as well as the le ve l of tr~nsgelll' expression; this was ,,1<0 the case for ti laggrin and I F a~,ociatioll in the itl /I ;(n, studies (Steinert el al. 19R I).

Expression ofProfuaggrin-Like Constructs Yields Granules Transicnt trallsfection of con~tructs containing- one or 111OI'e linker peptide seq u ence~ yie ld ed inllllulloreactive aggregates or illsolubil' protein. T his finding was surprising. but sugge~tivc of the lI ative epidermal dispositioll of protilagg-rin in keratohyalin. The reHllts indicatc that the lillker peptide it'clfinHlIencl'~ the belwvior of the expressed protein in the cell. The efli.'ct of the linker nUl)' he via fonnatiol1 ofintennolccular a~~ociatiollS with tilaggrin sequence> or by association between the h)'drnphnhic linkl'/' '<'qlll~nce, thenl'el\'e<. leading to aggrl'gation . Such an association might he (",p,'rted to he stronger in the FI AG-FG5 protcill (which contain< fivc lillkcrs) Ihall in the FLAG-FG I protl' ill , which has a singlc lill"er. EXl'l'I'ill1ellt,IlI)" the tive-dol1lain protl'ill (FLAG-FG5) ) ielded ollly !--'rmllll.,r slailling whereas rhe FLAG-FC I COllstJ'Ul't gave both gnJlular ,lIld diftil,t' staining. /11 /1;lm sllIdies demonstrate Ih,lt protl'olyric prnlildggrin fragrncnts cOlllainillg thn:e to five lilal:\gl;n dmllaills ,\1-': I<lrgl'iy insoluble ill aqueou< hutrer,. ",hnea< olle- to two-dlllll"in fra/:.'1nclI(s partition betweell thl' soluhk alld ill,,,IlIble li ':I('(iuII' (Re,illg (.( "I. 1995b; Thulin, I Y'>5). comi,tellt with 0111' ~ilHiillg\.

Profilaggrin is phnsplwrylated at lIlultipil' ,erille re<idlle< (Lonsdale-Eccles el ai, 19H2: R",ing ('I (/1, 19H5: I ()I).'),I) . alld phosphate has heen " l ~ge,('ed a. a potenlially illlpOl'lallt f:1l·tnr fil!'

the co rrect folding of I'rotil.lggrill alld SUb'l'lIUl'llI "n.llohyalill formation (Dale cl III. 1(94) . 11t' lIet,. an alternalivc explanatioll tor the aggregates in eel" e''1l1'es~illg prolilaggrill - like "()II'trun~ i~ that the prott!in is phmphorylarco. T\Vo-dimemiollnl gl'1 ,'Il'ctrophore­sis, however. showl·d that, although <tlllle pho'phnr,)'latinll or the ectopically expressed tilaggrin :lnel onl'- ;1110 tive- dolllnill protein<

J-II .A(;(;RIN I '\I'IU '~ION (, '\USI' 11 Cllil AI";1- 185

ma), occur. it is 1I0t a major tC:lture of the fiJaggrin proteins expre."ed in cu llu red ce lls (data l10t ~ho\\'n). We conclude that the formation of granules with protilaggrin- like constructs cannot be explained in terms of exten~i\'l' phosphor),lation .

Ectopic Expression ofFilaggrin Results in Nuclear Changes and Apparent Cell Death A dramatic tinding of this swd)' was that ftlaggrin also produced prol~)Und cffects on cell shape, which often coincidl'cl with disruption of thc nuc lt'ar membrane and los< of attachlllent to the cultllre di;h. These elfects were obser\'ed with both tilaggrin and profilaggrin-likc constru cts. Loss of cell viability ill filaggrin-positi \'e ce lls was shown by the condensation of cytoplasm and nuclens, and lifting of cells frolll the surface.

Fihlggrin toxic ity is also shown by ex pre. sion of both tllaggrin and the co-transfccted i3-gal,ll'lmidase in the nucleus (Figs 5; JA) . In cells expre<sing on l), /3-galactosidase. the immunoreactive pro­tein was never ohserved in thl' nuclells (e.g. , Fig 5C), indicating that overe"pres~io/l of the control protein ,,1"/1(' was not slIfficil'nr (() induce ch,\IIgc> in \Iuclc:lr integrity and IF organiz.ltion. Intranu­clear localizatioll of tilaggrin. hut not IF proteins, was obsel'\'ed 1'0

varying extents in ce ll , expres<ing all of rhe constructs tested. Filaggrin could disrupt the nuclcar melllbrallc illlegrity directly. by interactillg with nllclc:l r lal11ills, or indirectl),. by acti\'a ting apopro­tic pathwa)'(s) as ;1 resulr of C)'loskeletal dalllage alld cellular stre". The lIuclear lalllilla network , which Ii s on rile inner as peer of the nuclear envelope, consists largely of rype V lamin I F proteins (A chi CI

ai, 1986; reviewed ill Gerace and Burke. \ 988). Although filaggrin interacts \. ith mu ltiple IF proteins (Steinert ('I Ill . 19R I). its association with lamins has not been directly e\'aluated. In this stlldy, co­localization of blllin 13 and tilaggrin in sOll1e ce lls (e.g .. Fig 6C,D) ~ug~ests that I-ilagi-rrin can as>ociar,' with nuclear lanullS (a$ well as \\;th thc cyroplasmic IF nctwork) . This is consistent with the ohservation that filaggrin interacts with the conserved n-helical rod dOll1ain present in all IF proteins, indllding nuclear la111ins (Mack rl al. 19(3). Ne\'ertheless. tilaggrin did not eo-Iocalizc with depol)111erin'd lamins when the nuclear e(welope was dispersed (Fig 6B).

An altcmati\'e explanation ti)r nuclear changes i, that tilaggriJl indirectl) causeS the disruption of the nuckar ('In-l'iope hy activation of tile apoptotic p<lthway as :l result of a dal11aged cytoskeleton. The ccll 11Iorphology, including sh";nkage of the c)'ropla'111 ,l round thl' nncleus and nuclear condensation, is consistent with the occurrcnce of ,l!'O­

ptosis (fhr rcview<. sec Cohen. 1991 : H a;lke and Polakowska, 1 Y9J) . uciear envelopc dismption al,o occurs in ~poproti c cel"- coinciding

with degrad,'tinn of nuclear lamins to lowcr lI1olt'clIlar \\ eight peptidc< (Oherhanllller "I ai, 199-1: Ne;lInari CI III. 19(5).

Our tindings show ~o1lle similariries ro othel' ~tudies ill \\ hi r h gem's ellcodin~ 111 a1ll111.lIi:lll ,l1Id \' irus-l'lIl'oded I FA Ps. 0r 'pecitic d01llain< of IFAPs, were ectopically e"prl's'l'd in culrured epithelial n'l" in order to ,rudy rheir fUllctioll (reviewcd in Pn'<lancl. 1(96). Both de<lIlopl'lkill I and the rel,lted prmcin. plectin. ('ontain C-tl'1'l1linal sClJul'nCl's l'omisring of repeating domains of 176 .1111ino acids that co-aligll Wilh, and e\,l'ntll:1l1)' coll"p,e. th{' keratin and villlentin IF 11,'( works of epithelial cells illto perinuclear aggrq(.lte , (Stappl'lIhl'ck and Grel'lI, I ()92: StaPl'cnheck ('1 (/1, 199,,: Wiche ('I (//, I 99J). Collap'e ()fthe IF c)'t("kelt-ton also has heen rq)0rred for novel I F-associated (or di,rupting) protl·i n, ellrod,'d hy <evl'ral ()NA (1I11l0r viru,t'<, including the E-I protein of Cl'rtaill IlIl1nan papillmllavinl<l'~ (Ihlorb<lr rr (tl. \99\; Roherts rr 111. \993; \')94) .

ilaggl-ill, IWWl' \'cr. had :Idditiollal ('If,'cr< 011 ce ll shape. lIu cleal' ml' mbr,lIle ,t1'llctlll'e. and appart'nt ce ll \'iahilir)'. sug!,!estillg a t""it' effect. Ithough r('l llStrll ct< encoding L'ert~in plt-ctin (\'\fiche "I 111. I Q')3). E-I (Robert, ('I III. 19<)": 199-1), and 1,lillt"s (·yto\..l'I'atin p1'CHeim (Badl'l' ('I til. 19(1) were loc:di7cd to \'arying degrees in rhc nu c l,'u,. tht'n' was 110 apparent dfl'ct on 1I11c1car membrane il1ll'!~­rit y or cell \·iahility . Thl' lllll'stioll 1'('l11nil1s: dne' tilaggrin ('aUSt' IF collap,e. which rhcn re'lIlt·s in the lo s~ of nuclt':Ir illtegrit~ .lI1d I()l'alization oftilaggrin ill the lIUciellS or is tilaggrin to"ic to thl' t'ell Ieadillg to nuclear chJlIgcs ,11Id sub<equclll IF COIl'IPSt'? The tlrst IIl I'l'halli'm i, ,upporl'ed hy our req"t~ sho",ing IF c('llbp'{' millg Fe-L comtructs, as well :I, by tht' original ill ,,;11'1' .lg!!"eglltioll

186 DALE fiT r l L n IE .JOURNAl 01 INVI:S 11(;" IIVic I JERMA 1'01 OCy

Figure 6. Filaggrin ~xpre~sion disrupts the nuclear envelope. Transfected OS-7 cells. tixe.d and .Iabeled for double il11nlunoAuorcscence With po l )'c1o~lal antl-human hlaggnn (FG) wIth an FITC ~ccondllry and monoclonal antihody to nuelear lamll1 U Wlt~1 a 1" "x~' Red « 'cond.II·), (A-/-/) and o["e"'C(j by conlocal nucroscopy. The pairs «A) and (B). ~rc.) r"prc~c nc cwo V1CWS ofch" salllC field. (A-f) wcre lr;lmfcctl'd wIth thl' J"I A(;:-FGS <'onstron ; (E) and (To) wlch thc FG-L construct; and (G) and (H) wIth the allll-sense (FLAG-FG(as» construct. No!:e loss of Lill' nuele;,,' ellvelope III hlaggrlll-positiv" ells i (B) in which star-shaped aggregates arc stained with the lal1lin anribody (-+). T he ce ll in (0) show\ co- loca lization or libggrill- po\ ilivt· gHll ul cs with th: lIuclear envelope. The ce ll is (F) shows a co llapsed nucleus (-+). Non"," lam in staining is sec II in the anti-sense control (1-/) .

swdies; a toxjc effect of filaggrin cannot be ntled out at thjs time, however, and may be one effect of the profilaggrin-like granules expressed in the cytoplasm and Jluclells (sec Fig 4) .

Results of Expression of Filaggrin by Transient Transfec­tion Parallel Events of Tertninal Differentiation in Kerati­nocytes During the transition of the epidernlal granular ceU to the con-ufied ceU. the profilaggrin in keratohyalin granules is converted enzymatically to tilaggrin . wruch interacts with keratin IFs. Keratin IFs then bccome densely packed in the condensed cytoplasm characteristic of the stratum corncum . Our results offer strong support for the hypothesis that fi laggJ;n ajds in the dense packing of keratin IFs in the con-uiicd ceUs and suggests two additional important characteristics of fi laggJ;n and profilaggrin. First.. that the Ijnker peptide sequence contributes to profilaggrin aggregation jnto gJ·'\.I1ulcs, and therefore may be critical in preventing premature IF aggregarion, and second, tbat filaggrin (perhaps in combination with other proteins) may be involved in triggering cell death as it occurs during the termjnaJ differenriation of kerati.nocytes. It has been suggested that tenninaJ differentiation in epidennis constitutes a special fonn of apoptosis or programmed cell death (Fesus el nl, 1991; Haake and Polakowska. 1993). Many events occur at tl-us transition to yield the protective, fi.lllcti onal outer layer of the skin, including the loss of cellular organel les and the nucleus, and degradation of cellular DNA. CeUs in transition between the granular and comified layers have been shown to stain for fi-agJl1ellted DNA characteristic of apoptosis (Gavrieli et III, 1992; Polakowska ('I nl, 1994; Tamada el nl, 1994). In this study, tr'lIlsfected cel ls expressillg filaggrin show collapse of cell cytoplasm, disruption of the nuclear envelope, nuclear condensation, rounding up of cells. and loss of attachment, all features associated with pro­gJ'al1lmed cell death.

Our results support the role of filaggrin as an IFAP in livillg ce ll s and show that it leads to reorganization of both vimcntin ,md keratin IF networks. This functional test of the action of filaggri.n

~ 60 ]

50 u

" Z 40

a: 30 .. c

" 20

" c 0 10 0

FG-L. FG+L FLAG-FG1/5 AIIFG

Figure 7. Filaggrin expression resuhs in alterations in nuclear chrolllatin. COS-7 ("cll, tra",(cered with I-LAG-f(; I :I"d stailled with polyclonal allri-hunl3n til aggrill (FG) with a" 1-1 rc \t'colldary (A) and with DAPI stain for ])NA (H) "lid observed hy l'l'ilillol"C,celll'l' . Note rhe condensed nllclei ill the rd"ggrin posilive eel" (-0) . (C) S1I1l1111:1ry of allal),sis ofOAPI-,raillcd nuclei of COS ce lls c)(prc,sillg lilaggrill . The percelltage of abllorl1la1 or cOllden,cd nuclei is ,howlI Ii-,r n,lI, exprl'"illg the IWO t),pes of lilaggrin ('omtru t'. compared to cells tr.lnsfl'('ted with a cOlltrol (filaggrin anti-sense) COIl 'itruct. The r(,~lIh~ 'Hllnnlarl7C d~'t,1 fi'om ~cvc r;11 tran,fccrion cxperimcllt' . Bm" indio lte C I SI) . III cach data Sl·t. the difi"ercllce bctween scnse and anri-sell,e ("ons truct, j\ highl y \igllificallt (I' .." (1.1101).

VOL. I 11K. NO . 1' [' IJRUAlt Y 1')'17

gives strong support f()r its ro ll' in the packing of keratin I F~ in the differentiating ce ll s of the epidermis. Expression of"profilaggrin-like constructs containing linker selluence~ yield, protein aggregate~. w hile filaggrin itself i, (!etected throughout the n· ll ; both came IF network collapse and result in changes or cell sha pe ano 1m, of nuclear integrity, all fi.'aturt'S that occur during teJ'lllinal diffe renti­ation in keratinizing epithe lial tissues.

J"y·e wish /0 ''' ,wI.! Dr. jeHI'/,II C'trlTC,/f Jtr 'ftt ;lIl/o/llaill IlI'Ilm.'I"1 l'llismid, Iflllt't

KiI"ball JiJl' {'xcd/l'llt le(IIII;(ol assistrlll({', Bmb(lftl Hjl,t.!rl .Ii" ~ISS;S'II"{(, fI,,.,h rdl (I~/frm " Dr. Km"'" 51('1'"('IIS mill A Is. '-)IIId" T.t' ,!j"I/u' ,1/ •• /tnl/flf C;4"lI'(;n: C'"n' L",bomt0'1'.Iiw

DNA seqllrtlrillJ!. ",,,/ n/,. II 'il/in/ll eI//1"" JII' ,.,ili .. "II" """,Ii,~~ Ihi.' /II""l1sni/" . II ',· JI/'flfiflllly ,,(kll""''''({~(' 1/'" 11 './1 /. "" rk (,"'111", Ii"~ A""I//,n'" S'II'/i,''< /;'/' liS" ,,((,"/i,('(I/ m;croswpyIari/ifif's. 'r,,;.\' 'I'cwk ,/,,/,' SlIf'I',,/uti '/,r IV"tj",W/ Im-til lilfS' f~r H('a/;" (;'rmlfs

RJ7 DE-0466() "III/ !'O/ rltII-21557 r,,) B. A. D,,/,'). /~ . Il. I'''' 's /",,,/ i., t/,,· ""(i/,irtll of a Cm'err Dt',Jl'/ClI'"w"t A,p,ml.!;·c'''' ,ht' [)t"-"'11I(l/I~t!}' /:",,,,,Inri,,,,.

KEFEIU2NCES

Aebj U . Cohll J, Bllhlc I , ljt""lCC 1:'1 h I.' 1I11cll', lI' I,lInilla i:-o ,I IIInll'\ nrJ.. \.)1 illl e rlll c dl.lll'

rype liI:IIlH' IIt'i . Ntll"I'f J2.\:!)C/()-S(.4. I 'Hi(.

Albers K. Fuch, E: 'I'll,· 1I1 olL'l'1IIar hiology or intl'Tll1 C'di;lll.' IIlamelll prml.'in<;; . 1111 R,." Cy"" 1 3~ ; 2 4.l -27<). I ~n

Baden HP. Kuhilll:-O .I : The ~ro""1 .lIId d,II"t" ·l'lIli.llion til' .. :ullurl,d Ill'whorn r.n keral.inO('ytl·\, 1 I", '('si nr.,.",IIf,I/ XU ; 1 ,1~ - I ]0, 1')H.\

Bader BL, Magin ' 1 M. 1 ~ ll.'lIlk· nl1l ; 1I111 M. "'"11111'1' ..,. Fr;IItJ..t..' \V\V: Intl'nlll.'tii,Hc

filamellt\ l~mllt..'l1 de' I/, 1I'1t (1"(1111 Lui Il'"'' qwh'ralll1\ ill t!l\' \:~wp l.I\11l .Hld 111 thl' nucku< . .1 Cdl (!j,,1 I 11.1 ~'IJ-J.l1l7. I ')1) I

Carroll JM, Albl'r\ KM, G:1r1irk .lA, II<1rringl o ll It. T,lidllll .1I1 I B: 11\\l1l' ,lI1d ..:(r.IlUIll o;pecific l"prl'~ \il)lI 01 lhl' hllll1 :111 ,,1\ nltH nil prllJllflh.'r III Ir,IIl~p;l'llll' mll'C. J'nll

NIlII Arml Srj ( IS. I 'JI) : 1 1I271l-11127~. 1')')1

Ca.rroll JM, Taidllllull I B: ( ' II:rI',u: It..' ri/alioll ul Ihl.' 1111111;11\ ill\nlllCrllI I'lllilltlll' r u\in).! a tran,,:u.' 111 be!;1 gillartn,id.I:-Oc .1\\.1)" . .1 ( :,,11 .'\/1 I IJ'\ :I).!:;_ I).\O, 1 ()f),1

Cohcll JJ ; Prognlllll1lcd n· 1I dl·.JlII ill ihc IIIIIIIIIIH.: ~y "t.·'" . 111, ' 1"'","IIcll 5n:S:l-~5. I 91) I Dale UA. Gu", II AM. Ht..·l kllr:lll P. KiJl lh~llI JU ... Rc\il1g KA ' ( ·h.lr,1l'tl' ri7:IfIOIl oj" l\\n

m onoclolla l ,lIl1ihlldit- .. to htlmall l'pidcrJlI.11 kt·r.HPh }'.lIill : 1'l:'1( (i v it~ wilh liL1ggrin ~l1l d rL' b( cd I,rol(·ill": . II'IPI·,~I /)1 '11 11 ." .. 1 HH: \1)(1 - \ 1,\ . I f)H 7

Dale UA . Ilolhrook KA. S;Cilh'I'1 PM ; t\":\l'lIlhh ()r \11':1111111 (Onll' llIll h."11 pnll(' in .lIld kcmtin lilallH'lI1\ ill I11.H·rolihrik Stt(lIrf 27(1:1'2.')-7 .\ I , 197M

Dale I3A. Rl'\ il1 ~ KA, Prl.'\LI1H~ l-t ll: Kcratllhyalill j.tralltllt..· prt)l\'III"' . In : LdtJ,h 1M, LlIll.· ES. Wan I M (elk) /'III' J{l'rl1li"II,)',r II,mllllllllk , ( : 'IIIIIH"ldg:l' Ullivl..'l"\it) P,·c".

alllbridltc. U.K . • 1'!Y4 . 1'1' . . 121-.\';o Doorbar J. Ely S. -'h.- rling'. M, Il'a ll C. Cmwti)rd l ~pt'( ifl( ' Il1ll.'r.lctiOJl h .... tWCl·1l IIPV

16 E1 E4 .mel f)"wk,'.-alll l " rt..·,\ul" i n (o ll"l'\l' 0 1 I Ill' l.·pithe".11 4o' t.'11 1I11 (.'rmcdi:lIl' filar-nclI! IIcl\\ork. 8111HH' J=,2:H2~ -li.:!7. 11)'-' I

Eichner R. BOlliu P. SUIl r -'I : ( ' I:r 'i\i l il'.lIlon of ,' pldl' nllal L·J..ri.llIll<ri .11'"Cording 10 Illl' ir imnIUllOrC;(llivil),. 1\(1l' I(·t lric poilli . ,IIId I1Uldl.' (lll"prl'\~i(lll 1(.'('/1 HiII/'Hkl.\HH 1396. 19R4

Fcsu~ L. J)nvi c~ I~ . Pi:I('t' lllilJi M : Apul'to\i\ : IlIn ll'ntl.lr IIH.' lll,IIII\III'1 in prO)!r;1I1 111I l' d

cell deMI> . ti'"./ (,'<"11 Hj,,1 sc,: 170 - 177. 1'1'11 Flcckmll11 p. Dall' H/\ . Ilvlhrook KA : Pn)liLlggrin . n high lllol\.·l"ullll WCI!!11l prl'('ur":Il'

of filil ggrill ill hlll1l:111 l·pidcrJlli ... llId CUhlll'l'lI J..l·ra tillllq 1\.',\ • .1 I""rtf nelllJ,UII'

85:507- 5 I 2, I ')X, Foisner R . Wit-he G: IlIlt·I'IIH.'d, .ll t' li lall1l' llI :t"''illd .l1l'd pI'OIl'in..:. ( ' ,,1'1 ()1'''' ( :"" HIIII

3;75-H I. 199 1

Gan SQ. Mdlridl' O\V, Idler \V\V , M.II'ktl\.1 N. ~f""lIll'n PM : ()rg·illll /,lIiml . 'lnltttlf(.' .

and polylliorphh11l' (lflhl.· !l1J1l1:m profil . I ~J!"i ll J!l' Il ~, Hi,I( IU'lII;S"1' 29:9.t ,\2-'}.t40, 19911 Gavrieli y, Slll'rl ll :l ll Y. B~1l S-SA : ltil'lJllli r,I Ii<1I1 nfpl'ogr:lI11l1H'cI re ll dl' :llli HI .cil" v i<1

~pl'cific lilhl'ling (lr lIudl'M DNA (ra~lIlt·llraIIOI1..1 ( ''''I HIIII II Y:4')J-Sf) I. 11}1)2

Gerace L. Uurkt, U: hll1l' lhmnl organi7(llh.m of til(' 1111('11..',11 l'll\ dUpl'. ,'I""" I{,',' CI'II BioI 4:JJ5-J7~. I'IXH

Haake AR. Pnlnkow\k" Itlt : (\·11 clC.1lh h) .lpopIO\l\ III cphkrl11 ,l l hinlog~ .I /""/ '.(1

/)rr","I<,1 I 0 I : 107- I I 2. 1')9.1

Harding CR, Scol.l lit: Ili \dtiilll' firh prnrcill": (fil.lJ..tgrin'): 'lfiICIlII:11 :':HHf "")('Ii0l1,11

hctcrogclIl:' ily durill~ t' p iLll'rIlla l Jitfl'rCJlti :Hinll , .1 1\1/1/ mnl 17 () :(.51 -(17.l . 19H.\ Haydock PV. UIOllllllli"l C, nnllllb :lU~h S. 1>,IIt' !lA . Il olhl'nok I<A. Ill, .. klH.11I P.

Anti'icn!r\t.~ proJilag-W'ill R.NA lkl:1)s ,\lid dl' crC'a ,~H" prolil.)l'tgrin l'~prc'i\iof1 111ld ;'I J[cr~ ;/1 'tUm diif'"l'I'('nLi fllioll o( ral l.'pidt'I'I1I:t1 kl·nltIIHwVll· ... I /" "('.\'1 nnwlunl 1()1 : 11~ - 121, . 1'1,)]

Haydock PV. I)a ll' BA : ' 11 ll~ l'l'PC'liI IV" "'InH'lul'C \)flltc prolili1tn.~rill l-tCIH' :J~ d l' f11 01l­str:ucd u,Ioin g l·pidl.'nllal )1lflfil :I!-.atrill l DNA ,) Hitll (.'111'''' 2ft 1. I,:!S.2I1 - 1 2:i;.!;;, I')t{(l

Kouklis PD. lluttol1 F, hu.·h\ I .: Making-.I t'Ullllt..'llllHl : dll (· .. ·1 hllldill~ ht' lW('CII k.cl\uin i.l1tcnnedial(" filalllt..'lll\ .111(1 (IC'i"lIHl\(H II .l l proll'in\,.1 ( 'I'll Hitlt 127' 1114') - I I)()(). 11}"4

Laen1mli UK : ( ' I c'l\'a~t~ 01' ~C,Ut ttlr:11 protcin..- dUl-il11! ,III..' .I\\l·mhlv or thl..· hl':"! of baclcri(lpitagl' 14 . NIII",,'l27 : MHl - M~~. 11)7 0

Lon~dale-En' h' ''-J ll. ll'lh~ 1 1)( '. llalc BA : ( ' llar:1 (·lt· I"I /.III(HI 01 ,I plul\ph4.'r)I .llt..'ll f()fIll

of rile illl<.' rl1lcd l.lh' lilalllL' lH ,lg~rl'J.!;, lIillg pn1tclll "il ,lgf,.tl'in. Hi"dlf'lIIiW)' 2 1 :S<)4t1 -594H. I ')H2

Mack j\'<I, 'stl'\'CII At.. "1('II11'/'l PM : IIH' 11 1t' ..-11.1111 \11 I or illll'r.llllUI1 01 lil,l~gnll \\ill1 imcnrH'diatt' filnlll{·lIt\ . I hl' iOlllC 7ipper h~Pl1lhC\I ' .1 1\/01 IIhl/ .:!'\2:=iO- MI. 11)1).\

Mal1abc M. S:1I1chl'7 M, SIlII 1 r. I >.l ll· BA : lr1tl'r.U"11011 or lihl J.tw-in "iih kel'.Hil1

I'll t\l;(a~IN FXI'RI'SSION Ci\USr~ II (01 I AI'SI 187

hLiIl1l'III\ during- .td\, 'H1n~u "I:tg-l" 01 1101'111;11 hUII1.111 l'PUlt'rIllal ditft.'rClll1.It1()1I :lIld III iclHh)"u\j\ nllgaril>. DijftTC",""WII ·IH:-,",-,i ll. 1991

t.·;lIIl.Hi N. Il·rn.lIldt'7 A. \X ' li )!hl \. KU'II..'r J , MI( \ 'llk\·, 11.1 : l1l· ... ",.l ll.llinn nfbmill HI Ilf'('n'dl..' ~ \,hg,ulludl..'l.hUIII.11 DNA fra)!I11I..·I1I<llilUl in ,lp"I'I.nlit.: Ih~ IIH)C) I I.! , ,lilt.! j\tllall,d Ih~·t11/l(~ll.' II ut'l l'i //",1111/11 ,1/ I c)·I:J7HX-J74}S. 199;

(>lWI'II .11111I1er I ·A. IllJdll'l.~f! ... r K·. I rfhe lll C, I h..' fcnh.h Iwr H.., P.I\c1k .1 M : ('itr(lm;lIin

I'lHldl..'l1\,lIiulI dUl'lllg ;'JpOpro\I'i 1\ .1<T(llllpilnil'd b) dt'l-,'T.ILbtioll of 1.1 111 ill .A -t 13, v. ilhmit t.' lIh:rm·l'd :u '!ivalion of nil.1 "111.1\" . 1 ( "'II HIIII l.:!h :H27-NJ7, I ()'1.1

Pnl;lko\\'\k:r !tit. Pian'lIttni (\1. Hardl' " R . Cohh;l1l1h I A. Ila.lJ...l' AR:"\p0I'IO ~ l o;; III

IIIIIWIII \kill dc.·\clllP"WIII : nhllp"n~l'nl'\'" pcri(k·rm .• lIld \lel11 Cl'Ih.. /)n' /)," 1')'1 : 17(, - 1 HH. 1 <)~4

Pn·\l.md "It : 1t1l1.'rt1h:di:ul.' lilnnwlll - .I\\lH.·i,lCl·d prolei,,\ . 111 : r=, "('n Ii (l'd.). "r,nlt/ t',/lfIlt'

FiI'''//''//''', ,1A I Prl.'\\. Crl'cl1wich. C ·l. I ')<J6. in pn.'''\

Prl·\l.lIId RH. I 1.1) (hxJ.. PV. Hl'l'km.1II P. Nirlll1..:uJ...,dri \\- . Ihlt, BA: ChM.t("tt..'n7llIinn 0" till' hlllll .1II lTidl'rm;1 1 prntilaggnn ~t·nt..'. (i4.'IUll11i l' org:1J1I1JIIOIl ;"tlld Idt'lIl1li ­

CllHlI1 n( .111 \ - 1011 hkl' cllfl tJl1I hllHlil1~ dmn.HIl .11 ell(' .Imino Il'nllil1ll\ . I HUll (n,.,,, 2 (,7:23772-2.17~ J. Iq'll .

R .I\\ IIIII!\ f\ V. SUlCt IR, H.lrdill g- ('R. Un" "l'r pJ\ Slr.ltlll11 (·OMll.'um l1l(li"-Wn7.1Iio ll :1I thc lIIoh . .'l"lIiH Il·"l'I..I II1 1'l'SI J)C"""lhl' 10.\ :7,) 1-740 , 11)')1

Il.. l.'\illg KA. AI-I\I.I\\ IN. B1W1li..JlIl\1 C. I Ii: d'"lilll p, )).tll· BA : Il1d~rcl1dcnt I'l.'g:ul:uioll of Iwo 1\ lopl.l\lui\- 1"1'(l('t.· .. "'ing ~' .I~l· \ n( rill' IIllt'IIIICdi.Ht· fil a III t\ 11 I ;·'\\(K i.l tI'd

pl'oll'in lila~~'TH1 and rolc nfC.I! ·+ ill t il t' \l'l'ond 'icagt· . .1 Bioi CII"", 26H:1SIJCJ-2S 1,15, 199'\,1

Rl'\II1t! KAt 1).lll' Ii". \X .11 :-0 11 K 1\ : /Vhilupl(' copit' \ Ilf pllo..:pho')I.llcJ lil.IJotgnn 111 ('pidc.:rm.d prntila~!!,.j11 denhm"r:Hl'd h~ .1I1,iI) \', pi Iryptic Pl.'I'Ijcil'c; . H;Ilt/lfllll'ifl1' 24:4 1(,7- ·11 75 .II)H5

Rc\illg KA,.Iollll"Oll It"'. \ :thh l\A.: ( ' h .lnll· tl·ri7iltiWl OfprO I4.,.I"t' pnKt'\\lIlg \itt'\ dllnng

rou\('I'''Ij.ul ofral pn,nJ.II!1--'l'ill 10 til ll~lT1ll . B""I".w;.mr J2 :1nU.\6 - lfl04:'. 19'1.'h R(,..:illg KA. Johl1\(lIl ItS. \Xlalsh I<A: M,I\~ \l'l·('tnlf111.'crh ,111 :lIY'1\ ol"':!1 pho"phnn.l.l ­

li(11I \itc\ III the illlern .1i rCpl.' .11 of 1"11 prnlilagg-rlll . plt'ellnor of .111 lIlft..'rnll'dl.-Hl'

lil.II11l'1H .liO'l.'tl.Hl'd protein Hh li lr .. 11I1 .• ,r)' 3 .. L(1I 77-"4X7. IW)5il

Itl''iilll4 K " . 1 hulill C. \X.' hitin~ K. AI- A!.I\\ I N, ~(l'I ,l d S: Char.It'lcriTari~ln ot I 'n)fi l.l~ ~rill l'ml()pnHcill .l\l· I , A r~g(1I ,llt~ d n lopl.l\lni, · t' lldt)prOI(' lna\ .. · I,.l f CI'I­dl.·f111I\ .• 1 Hi,,1 (:1".111 .! 7ll :.!S II) .\-..! ~ II)X. 1 C)I':;h

Rl'\lIlg K J\ . \X ',I"" J,;. •. \ , II.HI !!CIl -~ .. nfil·ld.l. n.tll· B" : Idl' lllllif,lli"11 of pnl(l'nl~tir dt' .1\ ,I!!l' \itt' \ III IiH~ 1'1l1I\ l.',",ion HI Pl'lltil.ll!g-ril1 In ftl.l~grill in 1I1,ll11l11ali.Ul

l' l,idl.'nni\ .1 Bhl/ ( .'/h·m lh~:ltU7-IS4';. I"~I)

Il.ohl..'ll" ~ . A,llIlIoiL' I, Cib\oll 1.1 . Rookl.· .. SM. Banoll (;.1 . t..; ;11Ii1l10n· I'll : MUI.lntHUI

,lIlaly'i'\ ol ' hum,1I1 papilllHll:l\'iru ... r ~ prt"l.'l11" : idt·lIl1lk.1l101l llr"'lrllllllr.11 f,',HUl'l' \

i l1lpon:lIl1 ill till' Innll.lIinll l,r C) Inpla\lllH" ' ·.4/n t<lkl'f:1til1 IIt·t\\'nrk~ 111 l'Pllhdl;11 \·clk.llmJ! (1~ :C, 4J 2- (1~4:; . 11)1)4

R'lhcrl\ S. 1\\1111101, ' 1. Jnhll~on (;1), KJ'l' h.k·r .1\:\1, C;,dIrJlH..lrt..· PII ' CIII.1I1L·Oll' ,lnd 111U l" ~l\;Jl hllm,lIl p.lpilllll11a\ irtl\ I ~ I'nlll'il1~ I(.rlll IIlll' nlll'dl ;Hl~ 111.llncnt lik,' \lrUl'lUrL'\ III l'pHhdlal cd!... IIIII/II\?), 11)7' 176- 1 S7, 19 j 1.\

S ;llllhl'4,.)(l",1 . 1 ril\, 'h r 1 . M.lIli.lll\ I . \/r,/I'III/III (.'1111';".1: I 1.,bflljl'llr,. \/"",,,,1 211d l'd

enId ~prill).! Il.lrh(lr 1 .lhnr.lI(\r~ Prl·\\. ('old Sprl11J! 11.lIh{ll' . Y. 19HQ. PI' I ft .(,h - Ih.(,7

~llIlth ~A. 1),11t' 11i\ ImmUlh)ll)gK IO,' .lli7,tllon nflil'It!IlTlII III 11IIItlJn ur.11 t..·pllh,·li.1 .1110

nnrcl.1linn \\ Irh "l'r,lIiuiz!lIl(lIl . J 1111 '1'(;' /)t''''''' lftl ~h : I h~-172. . 1')~6

!\1.11'1'(·lIht·lk 1\, HlInr..:i:lq!t..'r I:A. COI't'orall C 1. lUll 1111. \ ·ir.lla MI. (~rl't' lI KJ hlllt IInn.11 . 1Il .11~~i, lolf dl' \1II0pl .IJ...1II dOfll.llTl' "Pl'Clh.'.1I11111 01 lh~ 1I11Cr.Il.'"tt(\1l With "n,tlHl '·t'P',1.( Vi111l'lItil1 inh·nnt·di.ltl· lil:lllll.·nl Ih'I'\~lrk\ J Cdl Hilll 1 2,\:h\)1-70~. I<)V .

SI ilppcllhcd. I S. Cn~l'll ".I ' I Ill' dl' \lIIOpt.11...111 c,lrh,}\\ I l.l'rllllllU:-O l.· ll<1li~lI\ \\ Ith .111lt

"l't..'nfi c.llI) dl\rUI'l' il1tt'nnl'dl,Jlt.' lil;lI11l'IH I1l·t\·\ o l'k ... \\ hl'll l·'l"·l'\~l·d ITt nJ!rurl·d "'11 , . .1 1'1'11 11",1 I I II . I I (17- 12111). I qlj2

"'Il·innl PM. C;JIHIt'd I". rl'lkr I)C. I (lll\tbll'-fl"de\ rn , I )"It' !lA , Charill"h'n7;lIicln ur

.1 1'1.1\\ n( l':lllOllir prml'in:.. Ih.1I "'l"'l' ('1I;1";11I) lJlIl'~act with il1tl'rml·dl.ltt..' til;II1WIlI \,

/"1 )' Slit! IfllIl S" ( .'i -I 7H: '"97- -.l1 () I. I t)S I SIl'illl' rt P"'. M .lrl·ktH I.N : rill' I'rOI(' III" t'l.llill, 1J1.1}'!I,tl'm. kf.'rarin 1I1(I..'nll~di3te

lilnml.·Jlt ... l(lrTl,.'rlll. ,Ind \111;,11 prohllt' ridl pnllt·1I1 .. I ~ l1d 2 art..' l"'OcllPl'l' t itit' tT(H; iO IiIlJ..ed (Ol11l'ol1('n l \ ,,(tht' htllllrll1 l..'l',dl'nll.lll·lll'nilil'(i I'f'lIt'II\C'lopl..' . I Hlill r J,cw 271l: I 771l2- 1 77 1 I , II)<)S .

!l(l'\' (..·n ~\C. Ij"hl.' r "". ROt,)!' PRo SICllh'rt I'M : Biu",nlitNic pati", .. y, oftil.1i!p:nn and Innt' rIn- cwo 1H,I.lor prOll' lth l" prl' \'l'd h\ 1(' II1)1Il.1Ih dltrl.·rl·I1t1~Ilt.'ci l ' plde rJlul

J..cr.lll1lnqll'\ . .I .\,I1I1f HiI)/IO" . It;O - I61, II)f}O

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