Expression . of Anionic Sites at the Dertnoepibolic Junction M oto mu M anabe, M .D. , Shiga ku Ikeda, M .D ., T sutol11u ' Muram atsu, M .D., and H idco ki Oga wa, M.D. , Ph. D. Depart m cn ts of De rm ato logy, Jun te nd o Un ivers ity Sc hoo l o f M edi cin e (MM , SI, H O), To kyo; and N ara M edi ca l Uni ve rsit y (TM) , NaTa, J apan
T h e emcrgen ce of ani o ni c sites durin g base m ent m em brane zone forma ti on was studi ed usin g mi gratin g epid ermis in organ cui tu re as a m odel systcm . U lt rast ru ctu ra l in ves tigatio ns Ll sin g a stro ng ly ca ti o ni zed
probc revea led th at th e hepa ritin ase-s1ensiti ve, ani o ni c sites w ere fo rm ed syn chron o usly w ith th e ne wly built basal la min a after 7 days in culture. J II/ ves t D erlll atof 88:94-96,
ons ide rable ad va nces have bee, n achieved in th e analys is of th e loca li za ti o n of co ll age no us and no n co llageno us co mp o nen ts o n sk in base m ent mem brane zo ne (sB M Z) (reviewed in 11 -3]). H eparan sul fa te proteoglyca n (H SPG) is a peri cellui;ir m acro m o lecul e co nsisting of a co re pro tein bea rin g hepa ran s ul fa te g lycosam in og lycan chains, Jnd was fo und in m an y tiss ll es as a co mpo nen t of the base m ent m e m brane as we ll as of th e cell sur f:tce (rev iewcd in 14J). An tibod y prepa red aga inst H S PG isolated fro m the Engelb reth- H o lm-Swarm sa rco m a m atri x was fo und b y illllll uno Ru o rescence to react w ith sB M Z 15]. T his anti geni c distrib utio n was co ns ide red to co rres po nd w ith ul tras tructurall y de lllo nstrated ani o ni c sites alo ng th e epid erm al and derm al ed ges o f the basa l lamina (B L) [6]. In this locati on , H SPG pla yed a m aj o r ro le in th e reg ula tio n of permeabili ty b y crea ti o n o f a cha rgese lective ba rri er (rev iewed in [7]). Recen tl y, it h as been ~ e p o r ted th at Sch wa nn cells sy nth es ize 2 major H SPG that diffe r in size and ap pa rent distribu tio n. FurtherJ1lore, the large r (B L-associated) pro teoglyca n accu mul ates o nl y w hen Sch wa nn cells are acti ve ly sy nth esizin g B L and th e acc uillula tio n of the sm aller (m e mbrane-associated) pro teog lyca n is indepe nden t of B L produ cti o n [S]. N everthel ess, detail ed information relat in g to th e synth es is of H SPG d urin g sB M Z fo rmation has been rather sca rce to date. In th e present stud y, the exp ression of an io ni c sites, parti cul arl y in relatio n to that o f th e B L, was in vestigated usin g mi g ratin g epide rmis in o rga n culture (epiboly).
Ultrastructural and Tracer Studies The specim ens we re rem o ved frOIll th e culture afte r 7 d ays and we re stain ed acco rdin g to th e m eth o d repo rted pre vio usly usin g stro ng ly ca ti o ni zed po lyeth y leneimin e (PE l) as a trace r 16]. Fo r enzy m e di ges ti on stud y, th e specim ens we re di ges ted by 50 units/ IllI heparitin 3se in 0. 1 M sodiulll acetate bu ffe r (pH 7.0) at 43°C fo r 2 h and were sta in ed b y th e sa m e m eth od. After ro utin e processin g, ultrathin sectio ns w ere o bserved in a J E M 1200EX electro n mi crosco pe.
1987
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R ES ULTS T he BL fo rm ati o n alo ng th e der m oe pibo li c jun cti o n lagged far behind the ad va n cin g tip of th e mi g ratin g epidermis. T he ani o ni c sites co uld be vi suali zed in bo th the derm al and epiderm al ed ges o f th e continu o us area (Fig 1a) and end poi nt (Fig 1b) o f th e regenerated BL as sm all particl es (approxim ately 20 nm in di am eter) occwrin g at reg ul ar di stances fro m each o th er w ith a cente r-to- cen te r spacin g of approx im ately 60 nm . T hey we re rem o ved co mpletel y b y di ges ti o n w ith heparitin ase (Fig I e) . T hese res ults we re identical to th at of no rmal sBM Z repo rted previo usly 16]. Ad d itio nall y, th ey we re p resen t o n th e focal area of regenerated B L s ubj acent to th e he mid es m oso m e (Fig l li) . Occasio nall y, th ey co uld also be detected o n th e cell sur face of basa l cell s th at res ted o n de rmal g ro und substan ce and co llagen w itho ut an intervenin g B L stru cture (Fig 1e). H owever, the distan ces betwee n partieles were rath er irreg ular and th e size and electro n densit y w ere redu ced co mp ared with th e co ntinu o us area of regenerated BL, altho ug h it was uncl ea r w hy th ere we re such di fferences.
M ATE RI A LS AN D M ET H O D S Orga n Cul t ure System O rga n culture of adul t human skin was carried o ut acco rd in g to the m et ho d of Hintner et al [91. T he specim ens we re ke pt at 37°C in a humid atmos ph e re co ntaining 5'1'0 CO 2 in air fo r 7 days.
D ISC U SS IO N T he distin ct co mp o nents of sBM Z have been no ted to re-fo rm Lltili zin g orga n culture as a m o del sys te m . Hintn er et al repo rted th at bull o us pe mphigoid anti ge ns e merged sy nchro no usly w ith th e ad va ncing tip o f the mi g ratin g hum an epiderm al cells up to 7 d ays of culture, w hereas ty pe IV collage n and laminin app ea red w ith co nsiderable dela y linkin g to each o ther [9J. Stenl1 et al also repo rted th at in a 4S-h culture o f m o use skin , mi g ratin g e pide rm al cells co ntain ed typ e V co ll age n but no t ty pe IV co ll age n 1'101· In t his stud y, we have so ug ht to electron mi croscopi ca ll y examin e th e fo rm ati o n of ani o ni c sites in 7-d a y cultures of no rm al hum an skin , in o rd er to bette r und erstand th eir rel ati o nships to th e BL fo rm ati o n . Ult ras tru ctural studi es usin g a stro ngly cati o nized trace r (PE l) dem o nstra ted th at th e sm all PE l-positi ve
Ma llllsc ript received A pril 8, 1986; accepted for publi ca ti o n Jul y 8, 1986. Rep rint rcq ues ts to: Hi deo ki Ogawa. M .D . , Depa rtm c.1t of Derm atology, Jun tendo Unive rsity Schoo l of M edi ci nc, Ho ngo 2-1 - 1, 13 un kyo-k u , Tokyo 11 3, J apan. Abbrev iatio ns: BL: basa l lami ll a H SPG: hepara n sul fate proteoglyca n (s) PEl: po lycth yicncim in e s13MZ: ski n basement me m bra ne zo nc
0022-202X/87/l1OJ .SO
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(a nionic), heparitinase- sensiti ve particles were present o n the co ntinuous areas , end po int and fo cal area of regenerated BL, and basa l sur face w hi ch rested directl y on the dermis without BL. Since it has been reported that m ous e mammary epithelial cells deposit at their basa l surf.1ce :111 extra cellubr HSPC that binds to ty pe I coll agen "Il 'j and that type I co ll age n fibril s ca use these cells to accumulate a BL-like la ye r 11 2, 13] , our findin gs mi ght sugges t that the an ioni c sites were form ed synchro nously with BL formation , and that th e intera cti o n between the anioni c sites and dermal cO lllponent(s) wo uld be important durin g sB M Z remodelin g . There w as ab und ant ev idence that keratin ocy tes synth esized severa l sBMZ components (reviewed in [14]) includin g sulfated g lycosa minog lycans [1 5, 161. In o ur present stlld y, howeve r, it remained unclear w hether keratino cytes synthes ized BL-associated HSPC or w hat factor(s) were essenti al for HSPG biosynth es is. It wou ld be interesti ng in future research to determine w hi ch cell type is produ cin g the anioni c sites, and what other base m ent m embrane co mpo nents intera ct fo r sB MZ format io n. We "'ish ro r!wllk Mr. Mi rsllraka Yos hida alld Mr. Karsllhiro Sa'" (Cellrm! EM Lab , Jllllrwdo Urrillc/'siry ) for rlr e;/, rCc/llrien! assisrallcr.
AN ION IC SITES IN E1'I130 Ll C SU IU' ACE
95
Figure 1. E lectron mi c r og r~ph s o f dcrmoepiboli c junction stained w ith a ca tionic tra cer, polyethy lcneimin e (PE l). The PE l-pos iti ve p ~ rtiC\cs we re present on the collti nu ous :lrc~ s (a) and end po int (b) of the new ly synthes ized ill and we re removed after h e p~ritina sc di ges tion (e). They we re also present on th e foca l are~ o f the new ly sy nth es ized Bl subj acent to the hel11id es l11 oso rlI C (d) . Occas io nall y, sm aller particles o ccuring at irregular di stan ces with low electron den sity we re present on th e cell surface o f basa l cel ls whi ch rested on th e dermis w ith o ut BL (e). Arrowhead s indi cate PEl = positi ve particl es, Bl = basa l lamina . Bm's == 0.2 Mill .
REFE RE N CES I.
2.
Bri gg~ m~n
RA : Biochemi cal co mpos iti on of th e cpid er·t1lal-dert1l al junction and othcr bascm cnt membran es. J In vest Denn atol 78: 1-6, 1982 Stanley JR, Woodle y DT, Katz SI, M artin CR: Structurc ~ lld tlm ction of basc mcnt mcmbranes. J In ves t Dc rm~tol 79 (suppl):69s-72s, 1982
3.
Katz SI: The epidcrma l base ment /l1 c mbr~n c zo ne. Stl'll cture, o ntogcn y, a/ld role in di sc~ s e. JAm A ca d Dcrm~tol 11 : 1025- 1037, 1984
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C ~lI ag h c r JJ , l yo n M, Stew~ rd WI' : Structurc and fun ction o f he-
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Ha ssc ll In., Ro bey PC, Ba rrach H-:J, Wilczek J, Rcnn ard SI, Martin C I( : Iso latio n o f" hcpar, n su lfatc-co lltainin g protcog lyca n frolll basc /1lcllt mcmbran c. Proc Natl A c~d Sci USA 77: 44 94-4498, 1980
6.
Manabe M , O gawa I-I: Ultra s tl'll c tllr~ 1 dem onstration of ani oni c sites in basc mcnt mcmbran c zo ne by c~t i o ni c pro bes. J In vest Dcrm ato l 84: 19-2 1, 1985
7.
Kanwar YS: Bio ph ys io logy of glo merular filtrati on and proteinuri a. lab In vcs t 5 1:7-2 1, 1984
8.
M c ht ~
paran sulph ate protcog lyca ns. Biochcl1l J 236:3 13-325 , 1986
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MANABE ET AL
by Schwann cell of basal lamin a and membrane-associated heparan su lfate proteoglycans. J Cell Bioi 101:660-666, 1985 Hintner H, Fritsch PO, Foidart J-M, Stingl G, Schuler G, Katz SI: Expression of basement membrane zone antigen at the dermoepiboli c junction in organ culture of human skin.] Invest Dermatol 74:200-204, 1980 Stenn KS, Madri JA , Roll FJ: Mig rating epidermis produces AB2 collagen and requires continual coll agen synthesis for movement. N ature 277:229-232, 1979 Koda JE, Bernfield' M: Heparan sulfate proteoglycans from mouse mammary epithelial cells. Basal extracellul ar proteoglyc;in binds specifIcall y to ' native type I collagen fibrils. J BioI C hern 259: 11763-11770, 1984 David G , BernfIeld M: Collagen reduces glycosaminoglycan deg-
TI-I E JOURNAL OF INVESTIGATIVE DERMATOLOG),
radation by c41tured mammary ep ithelial cell s: possible mecha, nism for basal lamin a formation. Proc N atl Acad Sci USA 76:786-790, 1979 13. David G, Bernfield M: Type I co ll agen reduces the degradation Of basal lamina proteoglycan by mammary epithelial cell s. J Cell BioI 91:281-286, 1981 14. Prunieras M , Regnier M, Fougere S, Woodley D: Keratinocytes synthesize basal-lamina proteins in cultu re. J Invest Dermatol 81 (suppl):74s-81s, 1983 15. King IA: C haracterization of epidermal glycosaminoglycans synthesized in orga n culture. Biochim Bioph ys Acta 674:87-95, 1981 16. King lA , Tabiowa A: The dermi s is required for the synthes is of extracellular glycosaminoglycans in cultured pig epidermis. Biochitn Biophys Acta 632:234"':243, 1980