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

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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 0022-202X/95/S09.50 • SSDI0022-202X(9S)003 19- G • C opyright © 1995 by T he Sociery for In vestigative Dermatology. In c. 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.
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