Skin Fibroblasts Are the Only Source of Nidogen During Early Basal Lamina Formation in Vitro

Skin Fibroblasts Are the Only Source of Nidogen During Early Basal Lamina Formation In Vitro

Raul Fleischmajer, Alan Schechter, Marco Bruns, Jerome S. Perlish, E. Douglas MacDonald, Te-Cheng Pan,* Rupert Timpl,i" and Mon-Li Chu* Department of Dermatology, Mount Sinai School of Medicine, New York. New York; *Departments of l3ioclwmisrry and Mokcular Biology, and Dermatology. T homas .J efferson University. Philadelphia, Pennsy lvania. U.S.A.: and the "t Max-Pianck In stitute fo r Biochen1istry, Munich , Gcnnany

The purpose of this study was to determine whether choring fibrils. The cell origin of nidogen was deter nidogen, the linkage protein of the basal lamina, is of mined in the dennal model and in the epidermal and epidermal or dermal origin. The development of the dermal components of the keratinocyte dermal basal lamina was studied in an in 11itl'O skin model. model. Specific antibodies and a eDNA probe for Preputial fibroblasts seeded onto a nylon mesh at nidogen were used for immunofluorescence micro tached, proliferated, and developed a rich extracel scopy, Western and Northern blots, and for iu sit11 lular matrix (dermal model). Preputial keratinocytes hybridization studies. Our data show that fibroblasts were added to the dermal model to forn1 a keratino are the only source of nidogen during early basal cyte dermal model that ultrastructurally resembled la1nina formation. Although fibroblasts can synthe in many respects hUJ.nan skin. Ultrastructural analysis size nidogen and deposit it in the dermal matrix, no , revealed early stages of dermal development, includ basal lamina will form unless they are recombined ing an inc01nplete basal lamina, aggregates of dermal with keratinocytes. This suggests that the epidermis filamentous material connecting to the lamina densa, plays a major regulatory role in the production and bundles of 10-nm microfibrils, formation of prema assembly of nidogen into the basal lamina. J Jm,est ture hemides1nosontes, anchoring filaments, and an- Dermatol 105:597-601, 1995

asal laminae are sheetlike supramo lecular structures guircd to form stable basal lamina structures. Fu rther studies that are deposited n ear ceUs in a polarized fashion demonstrated a m esenchym al origin of nidogen during the embry (epithe l.ial and endotheli al cell s) or surro unding the onic development of many organ s and its integration into basa l entire cell (muscle, fat. and nerve cells) fl]. Although lamina in the vicinity of epitheli al cell s that contribute lam in in and the functions of the basal laminae are not fully coll agen IV to these structures (8-LO]. T he general importance of Bunderstood, they appear to be involved in establi sh ing and preserv such cellular cooperation was shown with antibodies that inhibit ing tissue architecture, providing anchorage for adjacent cells, the laminin-nidogen interaction [ J 1] and prevent the fonnMion of controlling cell migration and invasion, participating in branching new basal laminae during kidney tubulogencsis and lung branching morphogenesis of epithelia, and regulating blood fi ltration, and arc [1 OJ. W hether such cooperations also exist during sk.in develop also in volved in receptor locali zation. T he major components of the m ent has not been examined. basal lamina are type IV collagen, laminin, heparan sulfiHc proteo Early transplantation studies with epidermal tissues suggested glycan (perlecan), and nidogen (entactin). T he exact molecular that keratinocytcs are responsible for the formation of the basal architecture of basal laminae is not entirely known, although there lamina [1 2]. There is evidence that kcratinocytes can synthesize is evidence that type IV collagen as well as laminin can form lami.nin (13,1 4], type IV coll agen [15,16), and perlecan (17), but homotypic polymers [2-4]. nidogen has so far not been studied. R ecently, a new culture system Another crucial element in the supramolccular assembly of the has been developed in which fibroblasts grown in a nylon m esh basal lamina is apparently the 150-kDa glycoprotein nidogen , develop a ri ch extracellular matrix [1 8, 19). When such dermal which consists of three globular domains and some connecting models were recombined with kcratinocytcs, there w as a rather strUctures [5]. Nidogen binds through these globular domains with striking reconstruction of the sk.in including an epidermis, basal high affin ity to laminin, co Ll agen IV , and perlecan , and thus allows lamina, anchoring zoJJe , and a dermis (19 - 21] . It is interesting that the formation of ternary complexes [6, 7]. T his was interpreted to laminin, type IV coll agen, nidogen , and perl ccm1 were locali zed at indicate that nidogen is the essential mediator for connecting the the epitheli al stromal interface and also in the dennis. T he purpose independent networks of collagen IV and laminin, which is re- of this study was to determine thl:' tissue o ri gin (epidermal versus dermal) of nidogen in this new tbTee-dimcnsional i11 11 irro human cul ture system and to show that dUt·ing early basa l lamina forma Manuscript received March 8, '1995; fina l revision received June 13. tion, fibroblasts arc the exclusive producers of nidogen . 1995; accepted for publication June 15, 1995. Reprint requests to: Dr. R.aul Fleischmaj er, Department of Dermatology, MATER.IALS AND METHODS Box 1047, Mount Sinai School of Medicine, One Gustave L. Levy Place, Culture Systems T he dermal model and the keratinocytc dermal model New York, NY 'I 0029. were grown at Advanced Tiss ue Sciences (La Jolla, CA) aud shipped to our

597 598 FLEISCH MAJER ET AL T H E JOURNAL OF INVESTIGATIVE DER.MATOLOGY I laboratori es o n nutri en t agarosc. Keratinocytcs and fi broblasts were isolated burg, MD) i11 si t11 hybridization protocol. T he eDNA probes were rlissolved from human neonatal fo reskin b y sequential trypsin and collagenase diges in O ncor hybridization buffer (50% fo rmamide/2 X SSC; O ncor Sciences) tion and then expanded i nto monolayer cultures [1 8,19]. Briefl y, to obtain in the p resence or absence of 1 00 ng of unlabeled h o mologous probe. A the dermal model, fi broblasts were seeded onto a nylon mes h in Dul becco's total of 5-10 X :1O'' cpm in a volume of 40 ~-t l was added to each slide. T he minimal essential medi um ( D MEM) s upplemented with 10% fe tal b ovine sljdes were coverslipped and in cubated in a humidifie d scal ed conta.in er serum and 100 J.tg/ml ascorbic acid. A feeding schedul e fo r 26-27 d was overnight at 45°C . Sections were washed twice in 2 X SSC; incubated for established, alternatin g DMEM at 37°C with phosphate-buffered sa line a t 30 min in 50% fo rmamidc, 2 X SSC at 45°C; and t hen washed once in 2 X 4- 8°C [22]. To prepare the keratinocyte dermal m odel, keratin ocyte cul SSC and 1 X SSC, respectively. After d ehydration with increasin g e thanol tures were grown in n1 o no laycrs in kcratinocytc scrurn-frcc n1 cdiwn concentrations, the slides were ajr dried and immersed in Kodak autoradio (G IB CO, Grand Island, NY). K era tin ocytes were then seeded onto the graphk emulsion (diluted 1:1 with deioni zed water). In all experiments, dermal m odel, submerged f or 5- 7 d, a nd then r aised to an air-liquid slides were exposed in the dark at 4 °C for 5 d b efore development. interface [23 Jusing D MEM supplemented with 5% feta l bo vine serum, 100 Northern Blot Total RNA was isolated from fibroblasts and keratino J.tg/ml ascorbate, 0.5 J.tg/ml hydrocortisone, and a cholesterol-1ich lipid cyte monola ycrs and 24 meshes of d ermal m odel. Twenty- fo ur meshes of supplement (S igma, St. Loui s, MO). Kcratinocytes were kept at the the keratinocytc dermal model were t reated with thcrmolys in (1 .2 mg/ml) air- liq uid i ntcrf.1ce for 2- 4 weeks. to separate the e pidermis from the dermis. Total R.N As were isolated u sing Antibodies and eDNA Probes Affinity-puri fied ni dogen antibodi es acid guanidinium tl1iocyan ate/phenol/chl oroform 130]. ll...NA samples were were prepared fro m m ouse EHS (Engelbreth-Holm-Swarm) t umor (24] , separated on a 1% agarosc gel containing 6"/.o fo rmaldehyde, transferred t o and antibodies were ra ised in rabbits. Antibodies against human nidogen hybond N m embrane (Amersham, Chicago, IL), and hybridjzed t o the were also obtained with a recombinant product [5]. T he probe fo r human eDNA p robe labeled with 32 P by ni ck-translati on. H ybridization was njdogen was a 4.0-kb eDNA, as reported previo usly [25]. ·w e al so used a performed in 4 X SSC (1 SSC is 0.1 5 M NaCI, 0.015 M sodium citrate), 1.2-kb eDNA for glyceraldehyde-3-phosphate dehydrogenase (26]. 0.02% fi coll , 0.02% polyvin ylpyrrolidone, 0.02% bovine serum albumin , and 1 00 J.tg/ml sa lmo n sperm DNA at 65°C o vernight. T he membrane was Microscopy Samples from th e keratin ocyte dermal models Elec tron washed to a fin al stringency of 0.1 X SSC, 0.1 'Yo SDS at 60°C, and then were fixed in Karn ovsky's solution for 4 h at room temperature, post-fixed film at - 70°C in the presence of 1 exposed ovenught to Kodak X AR and stain ed t'll bl oc for 1 h in 1 % for 1 h in fcrrocyanidc osn1iun1 tctrox.i dc, in tensify-in g screens. phosphotungstic acid fo ll owed b y 1 h in 3'Y., uranyl acetate 127]. Samples were dehyd rated and embedded in Spurr's res in. Meshes were then r otated RESULT S at a 45° angle w ith the poin t of each square-shaped opening aligned wi th the kni fe edge. Semi- thin and ul tra-thin sections were obtained in a Sorva l Ultrastructure of 2- and 4-Week Keratinocyte Dermal MT-2 b ultramicrotome and examined in a J eol EM 100 electron micro Model Cultures Fo ur-week d ennal m o d el cultures revealed a scope. connecti ve tissu e structure con sisting o f fibrobl asts in parallel arran gem ent sep arated by ch ambers with a ric h e xtracellular m a lmntunoftuorescence Microscopy Specimens of the dermal model and g collagen fibrils 3 0 to 8 0 nm in diameter, 1 0-nm th e kcratin ocytc dcrm altnodel were cut in to 3-4-nlm sq uares and frozen in trix, containin T iss ue Tech O.C.T. embedding compound ( Miles Labs, Elkhart, IN). micro fibril s, and mono laye rs of fibroblasts i11 the upper and lower Frozen sections 5- 6 J.tlll thi ck were placed on polylys ine-coated slides, fixed ed ges o f the specimen, as re porte d previo usly [20,21,3 1]. Two in cold acetone (- 20°C) fo r 5 min, and subjected to indirect immuno w eek k eratinocyte d ermal m o de l specime n s re veale d a well-o rga Au oresccncc microscopy as described previ o usly 121 ]. Specimens were nized epidermis and an ab sent o r incomplete lamina den sa, which cxmnin cd w ith ·a Nikon microscope c~1uipp c d with epiAu o resccnce illunli was presen t in areas w ith and witho ut he midesm osomes, su ggesting nation. Human embryoni c skins (about 16-1 8 weeks) were obtajned from that t h ese stru ctures pro b ably d o n o t precede the dev elo pment of above. Antibodi es were spontaneous abortions and processed a s described th e lamina d en sa. T he re w e re numero u s vesicles connecting to the used in concentrations of 0.2 mg/ml. Controls consisted of pure rabbit lgG cell plasm a m embrane f acing t h e extracellular space. The lower or scrunt fro n1 nonin1mutti zed rabbits. b orde r of the ke ratinocyte d ermal m o de l c ulture, in contact with Western Blots Twenty-fo ur dermal model and keratinocyte dermal the plastic s uppo rt, revealed a m o n o layer of fibro blasts in p arallel model meshes (1 1 X 11 mm) were extracted in 0. 15 M N aCl in 0.05 M arrangem ent, con sisting of elo n gate d cells w ith multiple pinocy s (phenylmethylsulfonyl Tris-HCI, p H 7.4, containing protease inhibitor totic vesicles and a well-dev elo ped r o u g h endo plasmic reticulum. flu oride (3 mg/1]. p-chlo romercuribenzoate (3 mg/1] , 0.5 mM i odoacet H o w ever, th e n arrow intercellular spaces revealed small am o unts amide, an d 10 rn M ethylenediaminetetra acetic acid) (28]. T he total extract ce of a basal lamina. was dialyzed at 4°C against water for 24 h and lyophil.ized. T he dried extract o f inte rstitial matrix a nd no eviden was dissolved in Laemmli sample buffer, and comparable amounts of Localization of Nidogen at the Dermoepidermal Junction dodecylsul fate-polyacryl material were loaded onto a 4% to 20% sodium and in the Dermis Indirect immtmo Au o rescen ce microscopy of amide gel electrophores is (SDS-PAGE) gel (Daiich.i gel; In tegra ted Separn the d ermal m od el sho w ed that nidogen w as strongly presen t tion Systems, Natick, MA). All samples were reduced with 2 -mercapto rather unifo rm distribution (Fig etbanol before electrophoresis. Electrophoresis was performed in 25 mM thro ug h o ut the e ntire space in a T ri s. 1 92 mM glycine, and 0.1 % SDS, pH 8.3 , at a consta nt current of 50 1a.). In the 2- week k e ratinocyte d ermal m o d el, where k erati.nocytes mA per gel. Nidogcn p resent in the extracts was identifi ed b y Western bl ot w e re in contact with th e d ennis fo r 2 w eeks, nidogen showed a analysis using s pecif1c a nti bodies described above. distinct Lin ear distributio n at the de rmoepidermal junctio n , al Protein b ands were transferred t o ni trocellulose, treated with antibody tho ug h th e rest of th e de rmis a lso sho w e d nidogen d eposits w ithin 125 and 1-protein A (NEN, Boston, MA). and exposed to x-ray fi lm at o r aro und fibro blasts n ear th e basal lamina (Fig l/1) . [n the 4 - week - 20°C. In a ddition to the dermal model and keratinocyte dermal model keratinocyte de rmal m o del, m ost o f the nidogen staining was no ted tial keratin ocyte and fi broblast m onolayers from the same samples, prepu at the dermoepidermal junction and in fibro blasts, whereas the rest source (Advanced T iss ue Sciences) were processed as described above. o f the de rmal m atrix s howed o nly little staining (Fig 1c). Fetal Iu Situ Hybridiz ation Pl as mids contairting t he eDNA subcloncs were human skin (1 6 -17 w eeks o ld) showed the nidogen restricted to di gested with the a ppropriate restriction enzyme to liberate the eDNA the d ermoepidermal junction, a ro und h air follicles and capillaries, in serts. After di gestion, eDNA inserts were purified by excision of sli ces of whereas it w as absent in tbe e ntire dermal m atrix (no t shown) . This low-melting agarose-conta ining DNA bands fo llo wing e lectrophoretic sep suggests that the presen ce of nidogen in th e m o d el dennis repre aration. T he DNA in low- meltin g agarose was then di rectl y labeled by sents a n initial transitory s tage in the f ormatio n o f the b asal lamin a. rand0 111 prim er CXtCils·io n tO a specific activity Of 1 QH Cptn/ J.L g using 33 P-dATP 129 ]. T he radi olabeled probes were t hen purified fic 01n unincor Evidence for a Mesenchymal Origin of Nidogen Three porated nucleotide either on Scphadex G- 50 or b y selective precipitation diffe ren t approach es w er e u sed to determine the o ri gin o f nidogen with ammonium acetate/ethanol (29]. in the i11 11itro skin m od el. W estern blo ts fo r nidogen r evealed a Frozen sections of dermal model and keratin ocytc dermal model were o ad band in the range ofB0-100 kDa in th e extracts o f the dern1al post-tlxed in 4% paraformaldehyde, dehyd rated with increasing concentra br tions of ethanol, a nd a ir dried. After r ehydration in 2 X sodium citrate/ m ode l, keratinocyte de rmal model, a nd in fi broblast m o nolayers sodium chloride buffer (SSC), the sections were t reated with 100 J.tg/ml of (Fig 2). T he presence of multiple nidogen fragm ents in the gels proteinase K (in 2 X SSC) for 15 m in at room t emperature, then incubated (mo lecular w eig h t o f nidogen 150 kDa) is due to endogenous with pre-bl ockin g agents l and II foll owin g t he O ncor Sciences (Gaithers- proteolysis, which i s particularly hig h fo r human nidogen . How- VOL. 105, NO. 4 0 T O UER 1995 BASAL LAMINA FORMATION IN VI TRO 599

0-M K-0-M Monolayer

220 kD-

100 kD-

2 3 4

Figure 2. Dermal origin of uidogcn by Western blot. Western bl ot using nidogcn antibodi es after separati o n of extracted tiss ue proteins in a 4 1Yo to 20'!/o SDS-PAGE gel. Lnn e 1, dermal model (D-M); lane 2, keratinocytc dermal model (K-D-M); lane 3, fib roblast monolayer; lane 4. kcrati nocytc nto n o layer.

as the exclusive source o f nidogen , in agreement with d ata o b served in m any developme ntal studies. T hese studies have initia.ll y Figure 1. Nidogen originates in the dermis. Indirect umnunoAuorcs sho wn a tr ansien t dep ositio n o f nidogen in the dermis of embryonic cen ce n1.i croscopy v..dth 11idogcn antibodies. n) D ennal model. b) Kc r ;~ tin o t·ats [33], in m ammat-y glands [34], and in reticulin fibrils of lymph cyte dermal model, 2 weeks. E, epidermi s; D, dennis. r) Keratin ocyte no des [35]. !11 siw hybridizati o n o f vad o us m o use embryo organs derm al model, 4 weeks. d) Dermal model sta ined with puri fied rabbit IgG. including gang lia, to ng ue, jaw, heart, liver, sto m ach , kidney, and Bar, 50 J.Lnl. lung [8, 1 0,36] also e mphasized a 1muo r m esench ym al o rig in of nidogen . Its final d estinati o n . ho w ever, is the epithelial and o ther ever, no reacti ve band could be o btained fi·o m the ke ratinocyte monolayer. T hese data strong ly suggest a m esench ym al localizatio n ofnidogen. N o rthe rn bl ot analysis detected an mRNA o f 6.0 kb for nidogen in the de rmal m o del. A fte r the rmo lysin treatment, the keratinocyte dermal mo del re vealed a similar mRNA transcript in 6.0 kb- the de rmal component, whe reas the epidermis was n egative. Extracts o f m o no layer cultures revealed nidogen mRNA in the fibro blast cultures, but it was not present in ke ratinocytes (Fig 3). a l11 s itu hybridizatio n studies o f the ke ra tinocytc dermal m o del specin1e ns sh owed that o nly the de rmal compo ne nt contained mRNA fo r nidogen, whe reas practicall y no nidogen w as present in the e pidermis, thus correlating wel.l w ith the N o rthe rn blot data. /11 situ h ybridizati o n o f the de rmal m o del showed that fibro blasts in the upper and lower mo no la yers also expressed mi~A fo r nido GAPOH- gen, although some staining was also noted within the de rmal matrix (Fig 4). D IS C USSIO N Several previo us studies have sho wn that the fo rmati o n of basem ent membran es is very often dependent o n mesench ym al-epithe li al 2 3 4 5 inter acti o n s, which arc particul arl y o bvio us during c mbi)'Onic b de velopmen t and could sti ll exist in adult tissues ["I 0,32]. In the present study, w e have used the ke ratinocyte de rmal m odel culture, Figure 3. Dermal or1gm of nidogen mRNA by Northern blot w hich mimics in many aspects skin de velo pme nt (1 8 - 21 ]. A analysis. T he epid ermis was separated fi-otn the dennis ol a kerati nocyte dermal model by thcrmolys in digestion. A 30-J.L g sample of to tal RNA in particul ar ad vantage of the m o del is the possibility to initiate each lane was electrophoresed on a formaldehyde-!% aga rosc gel, trans basem ent m e mbrane fo rmatio n by adding keratinocytes to a pre ferred onto nylon membrane, and hybridized with 32P-labelcd eDNA formed fibro blasti c extracellular m atri x, w hich all ows examinatio n probes for human nidogen (n) and glycerald ehyde-3-ph osphare dehydroge of th e early m o lecular events o f the process. W e have concentrated nase (GAI? DH) (b ). Lane 1, dermal model; ln11 c 2. keratinocyte dem1 al here on nidogen synthesis because of its functio nal versatility [7]. rn odc J. cpidcnnis; laue 3. kcratinocytc dcrn1 al ntodcl. dennis; lau e 4, Antib o dy studi.es and mRNA amll ysis clearly identified fibro blasts fibroblast monola ye r; la11 e 5. kcratinocytc monolayer. 600 FLEISCI-IMAJ Eit UT AL T H E jOUltNAL OF INVESTIGATIV E DErtMATOLOGY I ...

Figure 4. Dermal origin of nidogcn by ;, situ hybridization. T iss ue sec tions were in cubated overnig ht w ith a 33 P- Iabelcd eDNA probe for nidogen. n) Kcratinocytc dermal model. E, epid er . ·.-'.~.. ~:· ...... ·= . • mis; D, dermis. IJ) Dermal model. c) ,.;..!.,..... ····· · '· ... Control treated with excess unlabeled ·. 1 • 'o . '·,:- ... eDNA probe. Bnr, 12.5 nm. .. . '. ~ .-.· . ' : .·

.... '\ ·. . ;l~~:,"'~·~:II,IIIO•J."r"''~ ...a _ b· ·.

basement m embranes, as seen in adult organs. If this pmccss is 4. Yu.rchenco PO, Cheng Y-S, CoJognato 1-1: Luninin fo rms an independent prevented by antibodies that block the interaction between laminin network in basement membranes. J Cell Bioi 11 7: 111 9-.11 33, 1992 5. FoxJW. Mayer U. N isd1t R. Aumaillcy M , R.cinh:.rdt D. W icdcmunn 1-1, Mann and nidogen, then nidogen relocali zation and the whole develop K. Timpl R. Krieg T , Engel J , Chu M-L: Recombinant nidogcn consists or mental process arc in terrupted [1 0]. three globular domains and m cd i <~ t cs binding of l ~nninin to co U ::~gca t type IV. W ith the dcm1al model subst,·ate, we could show that fibroblasts BMBOJ 10:3137-3146, 1991 alone produce large amo unts of nidogen and deposit the protein in G. AumaiU cy M. Batt:tglia C. Mayer U. R einhardt D, Nisc ht R , Timpl R , Foxj\V: a rather uniform f.1s hion in the interstitial matrix. T he addition of N idogcn mediates the form a don for ternary complexes o f basement membrane co1.nponents. Kiduq /II( 43:7-12, 1993 kcratinocytes to the model induces a time-dependent redistribution 7. Mayer U, Timpl R: Nidogcn: a versatile bind in g proreiit of basement mem~ o f nidogen. After 2 weeks of i11 cubation, when most keratinocytes bratJcs. In: Yurchcnco PD. Dirk D, Mcclwm RP (cds.). ExtmcdluiM 1\flacn·.Y have aJready attached to the derma.! equi va len t, the interstitial Assclllbl)' aud Stm ctllre. Academic Press, O rlando, FL, 1994, pp 389-4 16 nidogen staining was reduced and in part substituted for by staining 8. T homns T, Dzindck M: Genes coding for hn scmcnt rn cmhrane glycoproteins at the dermocpidermal junction. After 4 weeks, the relocali zation l:uninin, nidogcn nnd coll ngcn IV nre differentially expressed in the nervous system and by cpithclja), endothelial, and mesenchymal cells of the mouse process has progressed further and resulted in a spot-like appear embryo. Bxp Cell Res 208:54-67, 1993 ance of remnant nidogen deposits in the interstitium, w hi ch 9. Dziadck M: flolc of huninin-nidogcn complexes in bascml! nt membrane forma suggests restriction around individual fibroblasts. A similar m assive tion during embryonic development". Exp!'ricu tia (in press) and transient-dermal deposition of nidogen was reported previously 10. Ek blom P. Ekblom M. Feckcr L. K lein G. Z hang 1-1 -Y. Kadoya Y. Chu M-L, M:1yc r U, Tim pi It: Role of nlcscnchymal nidogcn fo r cpithdial morphogen for embryonic rat skin [33 J, which indicated a similar relocali zation esis in vitr.o. Dc:l!elopw cllt '120:2003-20 '1 4, 1994 during normal development. This process is apparently completed II. Mayer U. Nischt R, Posch! R., Mann K, Fukuda K, Gerl M. Yarnada Y, Timpl in the skin of 16-17-week-old human fetuses which, as shown R: A sin gle EGF-likc mo ti f of" lamiaiu is responsibl e for hi gh :lf1ini ty nidogeu here, have nidogen exclusively in basement membrane zones. bi nding. BMBO j 12: 1879-1885 . 1993 12. 13riggaman R.A. Dalldorf FG, W heeler CE jr: Formation and ori gin of basal T he presence of keratinocytes not only triggers nidogen accu lamina and :111 chorin g fibri ls in :~d ult ski 11 . J Cell Bioi 8 1:384-395. 19TI mulation at the dcrmocpidermal junction, but also decreases nido 13. Stnnlcy J, Hawley-Nelson P, Yaar M, Martin G. Katz S: Laminin and bullous gen deposition in the interstitial matrix, as indicated from strongly pemphigoid ;mdgcn 01 rc dislim.: t basement membrane proteins sy nthesized by reduced antibody staining. T he first effect could be due to a supply cpithcli:ll ce ll s. J IIII'CSt Dt:mwtol 82:456-459, "1982 '14. Woodley DT. Stanley JR, R.cesc MJ, O'Keefe Ej : Human dermal fibroblasts of basal lamina ligands, by either keratinocytes or fibroblasts that syn thesize laminiu. J lll iJCsr D('nnatol 90:679-683, "1984 bind nidogen. T hese li gands were previously id en tifi ed as laminins, IS. Ali ta lo K, Kuismanc11 E. Myllyla R., Kii stala U, Askoselj:nra ara S, Vahcri A: collagen IV, perlecan, and ftbul.ins, and have been shown to Extracellular matrix proteins of human cpidcnnal keratinocyrcs and fe eder 3T3 integrate themselves into la.rger complexes through connections cell s. ) Cell Bioi 94:497-505, 1982 '16. Peltonen J , Jaak ko la S, Gay K. O ls en D. C hu M-L, Uitto J : Express ion of made by nidogen [5-7 ,32,3 7]. Preliminary data in our laboratories extracellular matrix genes by culwred lt uman cell s: locali zation of messenger have shown the cell surface assembly of collagen IV in the matrix RNAs and antigenic cpitopcs. A 11al Bioc/Jc'/ 11 178:'184-193, 1989 site of basal keratinocytes (in preparation). In this context, it is also 17. Marinkovich MP. Lundstnun G P. Keene DR, Du rgcson R..E: T he dermal of interest that the laminin y-1 chain contributes a high-affinity site epidermal junction of human skin contains :1 novel bminin vnriant. J Cdl Bioi 11 9:695-703, 1992 for nidogen binding [11] and is shared by most lamini.J1 isoforms 18. Naughton GK, J:tcoh L. Naughton llA: A phys iological skin model for " in vitro" except for laminin-5, which is a typical component of anchoring tox.icity studies. In: Gold berg AM (cd.). l lltcmn.til' t: lvlctlwds iu TCIJ."icology. llol. filaments. T he reduction of interstitial ni.dogen very likely cannot 7. Mnry Ann Liebert. New York, 1989. pp :183-I SY be en tire.l y explai.J1 cd by its accumulation in the basem ent mem 19. Trigli a D, Sh erard-Bra:! S, Donnell y T, Kidd I, Naughton GK: A three dimensional hwmm dermal model substrate for " in vitro'' toxicological brane. Kcratinocytes may there fore down-regulate nidogen synthe studies. In: Goldberg AM (cd.). } lftcn lllfillc Alldlwds i11 Toxico fo,f.!y. llt1l. 8. tvlary sis by a negative feedback loop or trigger proteolytic system s tor Ann Liebert, New York. 1991 . pp 35 '1-362 degradation. T he latter possibility is indicated by the high sensitivity 20. Contard P, Bartel RL, j acobs L. Perl ish JS. MacDonald ED , Handler L, Cone D. of nidogcn to tissue proteases, including several matrix meta ll opro Flcischmajcr R: C ulturing: kcratinocyrcs aud fibroblasL'i in n 3-dimcusional mesh results in epidermnl differentiation and fornwrion of a basal lamina tcinascs [38]. Such mechanisms undoubtedly will be of general anchoring zone. J l1111esr Dcnuato f ·100:35-39, 1993 relevance, and the keratinocyte dermal model substrate could be an 2·1. Flcischmaj cr R, MacDonald ED, Contard P, PerH sh JS: lmmunochcmjstry of a appropriate model for fu rther experimental approaches. kcr ~l t:ino cy t c- fibrob l nst co-culture model for reconstru cti on of human skin.J 1-fisroclu! /11 C)ltoc/t em 4·1:"1359-1366, 1993 22 . Sli vka SR., Landecn LK. Zeigler F, Zimber MB. Bartel RL: Characterization, .REFERENCES barrier fi.m ction and drug metaboli sm of an " in vitro '" sk in model. J Im·rsz Dcruwtof '\ 00:40-46, 1993 ·1. T impl R , Dziadck M: Structure, development and molecular pathology of 23. Asselinca u D, Bernhard B, Uaill y C, Dannon M: Ep idermal morphogenesis and basement membranes. ltll Re11 l::x p P(lt/w/ 29: '1- 11 2, 1986 iuduction of the 67kD kcmtin polypeptide by culture o f human kcratinocytcs 2. Tirnpl R.., W iedemann 1-1. va n Dcldcn V, Furthmayr 1-1 , Kuhn K: A network at the liquid-air interface. Exf' Cell Res 159 :536-539. 1985 model for th e organization of ty pe IV coll agen molecul es in basement 24. T impl R . Dziadek M. Fujimara S. Nowack H. W ick G: Nidogen: a new

membrane. Eurj Uitwhc111 120:203-2 11 , ·1

K , Deutzman R.. Timpl R., Uitco J: Human nidogcn: complete amino acid 32. Ekblo m P: Bnsemcnt membranes in development. In : R ohrbach DH. Timpl R sequen ce nnd structural domai ns d educed from cDNAs and evidence fo r (cds.). JHolen tlnr mul Cellular Aspt:t:ts l!fBa.H:m t:ut klt•wbrniH:s. Ac;1dcmic Press . San po lymorphism of the gene. DNA 8:581-594. !989 Diego, CA. 1993, pp 359- 383 26. Tso JY. Sun YH, Ka o T I-l , Reece KS. W u R: Isolati on and characteri zation of 33. 1-log;m fiLM . Taylor A. Kurkincn M. C uclunan JR: Synthesis ~m d locali zation of rat and human g lyccraldchyd c-3-pltOsphacc dehydrogenase cDNAs; geno mic rwo sulpharcd glycoprotein s associated with bascmcnr me mbranes :md the complexity and molecul ar evolution of the gene. N uclc:ir IJ cid.s Res 13:2485- extracellul ar matrix. J Cell Bit1/ ')5 : 197-204 . 1982 2502, 1985 34. \Varburton MJ, Mo naghan P. Ferns SA. Rudland PS, Pcrusinghc N , C hung AE: 27. Hulmes DJS, J csior T-C. rvtiJJ cr A. -Dcrthct-Colo minas C. WoUf C: Electro n D istribution of cutat.: l"in in the ba ~c m c nt membrane o f rhe rar rnamm:u) · gland. microscopy shows periodic structure in coll agen fibri_l cross sections. Pror N at/ Ev idence fOr a non-epitheli:tl origin. Exp Cl'il R t:s 152:240- 254. 1984 Acad Sci USJJ 78:3567- 3571. 198'1 35. Kramer lt.H . ltosen SD. McDonald KA: Basement membrane co mp o n e nt !~ 28. Paulsson M. Aurnaillcy M , Dcurzm:mn R.._, Tim pi R. Beck K, Engel J: Laminin- associ:1tcd with the cxtrilt:cllul:lr mar_ri x- o f th e lymph node. Cell Tissflt' Rt·s nidogcn complex: extraction with chcla tin g agents and structural character 252:3 67- 375. 1988 ization. Eurj Biochc111 166' 11 - 19. 1987 36. T lt Oitlas T. D ziadck M: Expressio n of collagen I (IV) l:l nlinin and nidogcn genes 29. Sam qrook J. Fritsch EF. Mauiatis T : J\floltx ulm· C loniu,t;: Jl LaiJMalt11")' J\.lal/ual. Cold in the embryonic mouse lung: implicati o qs for br:mching m o rph ogc n cs i ~. J\'lcrh Spring Harbor Laborato ry, Cold Spriu g Hnrbor, NY, 1989 De" 45:193- 20 I. 1994 30. C homczynski P. Sacchi N: Single-step method of l"tNA isolation by nci d 37. 13 a tt:tglia C, Mayer U . Amnaillcy M. T impl 1 ~: Bascrncnt membrane heparan g: uanidinium thiocy:mntc-phcnolchlorofonn extracti o n . IJ unl TJiocil cw 162: sul f:nc protcoglycan hinds ro laminin hy irs heparan sul fa te chains ;md to 156-1 59, '1987 ni dogcn by sites in the protein core. Eurj 1Jiorhc11t 208:359-366, 1992 31. Flcischmaj er R . C ont:ard 1'. Schwartz E. MacDonald ED. Jacobs L. Sakai LY: 38. M:t yer U. Mann K, Tim pi R. , Murphy G: Sires of nidogen cleavage hy protcascs Elastin-associated microf!l)ril s ( I 0 nm) in a rhrec d imcusio nal fibroblast involved in tissue ho m cosr:1 sis and rcn10delling. Eur J /Jiclc/tf /11 2 17:877-884, cul ture. J ltwest Dcr/1/ntcl / 97:638-643, 1991 1993