Biochemical Composition of the Epidermal-Dermal Junction and Other Basement Membrane

Reprinted from REVIEW ARTICLE

Progress in Dermatology a bulletin published by t he DERM ATOLOGY FOUNDATION Co- Edi tors: Robert A. Brigga man, M.D. Lowell A. Goldsmith, M .D. North Carolina Memori al H ospita l Box :.l0:10, Duke Medical Center Chapel Hill , Nort h Caroli na ~75 1 5 Du rham, North Carolina 277 10

Biochemical Composition of the Epidermal-dermal Junction and Other Basement Membrane

R oBE RT A. B RIGGAMAN, M.D. Professor of Dermatology, University of North Ca.rolina, School of Medicine, Department of Dermatology, Chapel Hill, North Carolina. U.S.A.

During t he last several yeaTs, our knowledge of the biochem of the internal leaflet of the p lasma membrane. To no filaments ical composition of the e pidermal-dermal junction and the course toward the attachment plaque but actually terminate in ultrastructural location of the pathologic reaction in numerous a relatively electron dense zone separated from the plaque by skin diseases that affect this area has increased greatly. In this a narrow electron lu cent zone. paper, our purpose is to review current information in these T he lamina lucida (intermembraneous space, lamina rara) is areas. The epidermal-dermal junction h as been reviewed pre an electron lucent ru·ea located between the plasma membrane viously a nd the reader i s referred to these som ces for a more of the basal keratinocyte and the underlying basal lamina. general a nd comprehensive review of the subject [1-5). Lamina lucida is approximately 20-40 nm in thickness and is First, a brief review of the ultrastructural m orphology of the amorphous in most areas. In the lamina Iucida subjacent to epidermal-dermal junction is necessary to relate the subsequent hemidesmosomes a special structme termed the sub-basal studies. The e pidermal-dermal junction i s more complex and dense plaque appears as an electron dense line beneath the more highly organized ultrastructurally t han the s imple linear hemidesmosome. In this area, fine filaments traverse the lamina "basem ent membrane zo ne" between epidermis a nd dermis Iucida perpendicularly a nd mesh with the basal lamina. These noted b y light microscopy. Three different epidermal cell types, filaments have been called anchoring filaments. basal keratinocyte, melanocyte, and Merkel cell, c omprise a T he basal l amina (lamina densa) is a continuous electron portion of the e pidermal-dermal junction wi th each cell type dense layer which runs pru·allel to the plasma membrane, presenting a somewhat diffe rent a ppearance at the junction. sepru·ated from i t by the lamina Iucida. T he basal l amina has B asal keratinocytes form the vast majori ty of the junction zone an am orphous fine granulru· appearance and, although some and will be t he only cell considered h ere. The epidermal-dermal what vru·iable in thickness and density, measw-es approximately junction b etween b asal keratinocytes and dermis can be di 30-50 nm. Reduplication of the basal lamina is relatively com vided, for convenience, into 4 ru·eas proceeding from epidermis mon even in normal skin and may result from remodeling of toward the dermis (Figure). These ru·e: The basal cell plasma the epidermal-dermal junction as a fu nction of cell detachment, membrane wit h its special attachment structures, hemidesmo migration, and re-attachment in this area. Reduplication of the somes; lamina Iucida; th e basal lamina; and a sub-basal lamina basal lamina a nd other junctional structm es may be markedly fibrous zone. exaggerated in a variety of pathologic processes that affect the The plasma membrane of basal keratinocytes is a t rilaminar junctional zone. Mru·ked reduplication of basal lrunina is the structure measuring a pproximately 7-9 nanometers (nm) in ultrastructural counterpru·t of the basement membrane thick thickness. Electron d ense thickenings stud the p lasma mem ening, for example, as noted in cutaneous lupus erythematosus. brane a t frequent intervals. These structures are termed h emi The subbasal lamina fibrous ru·ea is situated immediately desmosomes and are diagramatically presented in the Figm e. beneath the basal lamina a nd is composed of three different T he hemidesmosome is composed of an electron d ense area, types of fibrous structures: anchoring fibrils, der mal microfibril the a ttachment plaque, that is present o n the cytoplasmic side bundles, and collagen fi bers. Anchoring fibrils have a unique structm·e which consist of a central, asymmetrically cross banded r egion with fa n-like ru-rays of smaller fibrillar compo This work was supported b y Research Grant 2 HOl AM 1054G, National Institutes of Health. nents extending from both ends. T he epidermal end of the Reprint requ ests to: Dr. Roberl A. Brigga man, Department of Der anchoring fibril meshes with the basal lamina while the dermal matology, The Un iversity of N orth Caro lina, School of M edi cine, end extends into the dennis. Frequently, the dermal portions of Chapel Hill, NC 27514. adjacent a nchoring fibrils fuse forming a n interlocki ng mesh- 2 BRIGGAMAN Vol. 78, No. 1 work in the d~rmis below the basal lamina. Collagen fibers are distinctive component in the sub-basal lamina area. These sometimes caught up in this mesh-work or surrounded by loops consist of bundles of parallel fibrils which pass deep into the of connecting anchoring fibrils but are never directly connected dermis essentially perpendicularly from the epidermal-dermal to the anchoring fibril. The dermal microfibril bundle is a junction. The epidermal ends of these bundles insert directly into the basal lamina. Individual fibrils are morphologically identical with the microfibrils that are a component of true elastic fibers [6, 7]. Indeed, some dermal microfibril bundles have been traced to connections with morphologically distinct elastic fibers thus documenting a direct connection between the basal lamina and elastic fibers in the dermis via these structures [8]. Typical collagen fibers are the third fibrous component in the sub-basal lamina area. These are randomly oriented and are most commonly found as single fibers or occasionally in groups of several fibers. Collagen fibers do not attach directly to the basal lamina or to other junctional structures as yet identified.

BIOCHEMICAL COMPOSITION OF BASEMENT MEMBRANE WITH PARTICULAR REFERENCE TO THE EPIDERMAL-DERMAL JUNCTION Before concentrating on the epidermal-dermal junction, we will first review current knowledge of biochemical composition of basement membranes in general [9,10]. Basement mem branes are composed of collagenous and noncollagenous com ponents which are tabulated below (Table). Basement Membrane Collagens Current evidence indicates that the basement membrane collagens are distinct from the interstitial collagens and can be subdivided into 3 groups, designated type IV, type V (AB collagen), and 7S collagen. Type IV collagens

FIG Epidermal-dermal junction in region of a hemidesmosome. To Type IV collagen is composed of 3 polypeptide chains that noftlaments (TF). Attachment plaque (AP). Plasma membrane (PM). form a macromolecule with triple helical domains similar to Subbasal d ense plaque (SBDP). Anchoring ftl aments (A}. Lamina other collagens. However, the constituent polypeptide chains of Iu cida (LL). Basal lamina (BL). Anchoring fibril (AF). Collagen fib er type IV collagen are larger than other collagens and are disul (COL}. Dermal microfibril bundle (MF). fide-linked. In addition, nonhelical sequences constitute a larger

TABLE I. Composition of basement membrane Basement membrane Moi.Wt. Cutaneous localization Source Basement membrane co mponents (BM) (K) distribution Presence Ultrastructural Collagenous T ype IV collagens >400 Lt l (IV} 160 Mouse EHS sar coma Ubiquitous + Basal lamina a 2 (IV) 140 Kidney and ocular BM Pro a 1 (IV} 185 Pro a 2 (IV) 170 Fetal membranes T ype V collagens 300 a A 100 Fetal membranes Vascular, + ? a B 100 muscle, skin, muscle BM aC bone, lung 7S collage n 360 Mouse EHS sarcoma Ubiquitous + ? kidney, ocula1· BM fetal membranes N onco llagenous Bullous pemphigoid antigens Epidermal 20, 9.2 Skin Skin, other + Lamina Iucida Urinary 19 Urine sq. epithelial High mol. wt. 220 Epidermal cell culture Lam in ins Soluble laminin 800-1000 EHS sarcoma Ubiquitous + Lamina Iucida Subunits 220 440 Insoluble laminin EHS sarcoma ? ? ? Hepanin sulfate pro 750 EHS sarcoma Ubiquitous Leoglycan + ? Fibronectin 440 Plasma, Ubiquitous + Lamina Iucida Subunits 220 Fibroblastic cells Amyloid P -compo plus dermis Kidney Kidney ? nent ? JUNCTION 3 Jan. J982 BIOCHEMICAL COMPOSITION OF THE EPIDERMAL-DERMAL portion of type IV collagen than other collagens as indicated by have been designated pro-a 1 (IV) and pro-ex2 (IV) to indicate the glycine content which is less than the 1/3 dictated by the their precursor relationship to the smaller tissue forms that are Gly-X-Y helix sequence. Distinctive differences are also evident now termed a 1 (IV) and a 2 (IV) [26- 28]. It is believed that in the content of other amino acids and in the posttranslational these represent the major alpha chain-like components of type hydroxylation and glycosylation of type IV collagen. The ratios IV collagen although other minor components have been iden of 3- to 4-hydroxyproline, of total hydroxyproline to proline and tified but not yet characterized [28]. hydroxylysine to lysine are greater in type IV collagen and serve as distinguishing featmes. Type IV collagen also has a Type V (AB) collagens which is present as disac high carbohydrate content, most of Concomitantly, 2 groups of investigators [29,30] discovered 2 bound to hydroxyly chrides containing glucose and galactose new collagen chains, termed ex A and a B (or A and B chains) sine. from various tissues including human placenta, skin, muscle, and confusion has arisen from at Considerable controversy lungs, bone and cartilate using limited pepsin protelysis fol type IV collagen and its constitu tempts to extract and isolate lowed by salt fractionation to isolate the collagenous fractions. from basement membrane ent alpha chains and polypeptides These collagens have been termed type V collagen (AB colla udies have been fraught with many containing tissues. These st gen). Type V collagen share featmes with both interstitial very small amount of base technical problems including the collagens (Types I-III) and the basement membrane collagens. most tissues and their relative ment membrane components in The moleculm· weights of the chains (approximately 100,000 difficult problem has been the inexcessibility. A particularly daltons) and their resistance to pepsin digestion are similar to membrane collagens such very limited solubility of basement the interstitial collagens. However, their amino acid content is required for their solubilization. that harsh procedures are similru· to peptides derived from isolated basement membranes. the interpretation of the These procedmes have complicated They contain relatively lm·ge amounts of 3-hyru·oxyproline and studies. hydroxylysine and relatively small amounts of alanine. A and solubilization of kidney and Initial studies utilizing pepsin B chains differ significantly and aTe thought to be separate that the major collage ocular basement membranes indicated proteins. The molecular organization of A and B chains into chain (100 K) , 3 of which were nous component was an alpha AB collagen remains controversial. Some workers have found helix and designated ex (IVh [11- assembled into a single triple a constant 1:2 A:B ratio in extracted tissues indicating a struc 13]. tme of a A (a B2) [29,32]. However, others have not found this found greater heterogeneity in the Subsequently, others have constant relationship in different tissues [30,33) and have ac basement membrane. The degree of collagenous components of tually found the absence of one of the chains in some tissues, the studies with some studies heterogeneity has varied among indicating that each are distinct collagens composed of 3 iden reporting extreme heterogeneity of collagenous components tical A or B chains (a A3 and a B3) rather than being hetero from 25,000 to 205,000 daltons that varied in molecular weight polymers. Because of the wide tissue distribution of type V evidence has established the [14,15]. Recently, considerable collagens and since they ru·e not isolated specifically from of at least 2 distinct alpha occmrence in basement membranes basement membrane, some have questioned their basement represent different gene products chain-like components that membrane association [10]. Indeed, it seems likely that type V of type IV collagen [16-22]. Much of the apparent heterogenity collagens ru·e not exclusively basement membrane components. of pepsin sensitive sites can be accounted for by the presence However, immunohistologic studies have documented their within the polypeptide chains [16,22]. basement membrane localization [34-36]. Antibodies to A to this field was the discov An important recent contribution chain, B chain and type V collagen have demonstrated their tumor, EHS sarcoma, that ery of a transplantable mmine presence in a vm·iety of basement membrane including lung and resembling basement mem produces an extra-cellular matrix kidney. In the kidney, types IV and AB collagens ru·e co of the tumor is that basement brane [23]. A special advantage distributed in the basement membrane of tubules, glomeruli harsh solubiliza membrane collagens can be isolated without and Bowman's capsule as well as in the mesangial stock [36]. tion treatments required in most other basement membrane acid extraction of the tumor isolation procedmes. Dilute acetic 7-S collagen matrix has lead to the isolation of two collagenous polypeptide chains with molecular weights of 160,000 and 140,000 daltons. 7-S collagen was frrst found in the insoluble residue after These polypeptides have helical as well as nonhelical domains extraction of mouse EHS tumor matrix [37) and later isolated and exist in the matrix as a trimer with molecular weight over from other somces including placenta, lens capsule, and kidney 400,000 daltons. The chains are cross-linked by disulfide bonds [38]. This collagen is sepru·ated from other basement membrane and have an amino acid composition similar to basement mem collagens by its relative resistance to bacterial collagenase. Its brane collagen purified from authentic basement membranes amino acid composition is similar to other basement membrane [21-24]. Antibodies prepared against mouse tumor basement collagens except for elevated levels of cysteine and tyrosine. 7- membrane collagen localized to the tumor matrix and to a S collagen occms as a very stable triple helical molecule with variety of basement membranes in tissues, including skin, thus a large number of disulfide bonds connecting component chains. confirming the basement membrane distribution of these pro It has a rod-like configuration on electron microscopy and teins [21,25]. occw·s in 2 forms: lru·ge form with molecular weight of approx In biosynthetic studies, cultured cells from various sources, imately 360,000 daltons and a sedimentation co-effic i e~t of 7.2 including EHS tumor, produce and release into the media S and a short form with moleculal" weight of approximately collagenous basement membrane polypeptides [26-28]. Two 225,000 daltons. 7S collagen is composed of subunit polypep 7-S col major polypeptides (approximately 180-185 K and 160-170 K) tides with molecular weights of 25,000-27,000 daltons. other were isolated without harsh solibulization procedmes. The 2 lagen is immunologically distinct from interstitial t~:nd polypeptides differ significantly from each other in the peptide basement membrane collagens. Immunofluorescent llllCr?s~opy irms that Jt 1s an patterns produced by cleavage with cyanogen bromide or pepsin with a specific anti-7 -S collagen antibody conf [38]. and are believed to represent distinct gene products. In the case abundant component of most basement membranes genous glycopro of EHS tumor cells [26], evidence indicates that the larger (185 · Noncollagenous glycoproteins: Noncolla of basement K and 170 K) polypeptides are precmsors of the smaller tissue teins and proteoglycans are significant components forms (160 K and 140 K) as a result of in vivo processing. membranes. antigens Evidence supporting this is that antibodies prepared to 160 K Bullous pemphigoid antigen: Bullous pemphigoid glycoproteins or 140 K chains precipitated the respective larger peptides. In (BP-Ag) ru·e noncollagenous basement membrane with bullous pemphigoid addition, similar polypeptide fragments were found after cleav that ru·e identified by their reactivity . Surprisingly, age with cyanogen bromide and pepsin. The 1m·ger peptides antibodies derived from patients with that disease 4 BRIGGAMAN Vol. 78, No. 1 a variety of materials have been recognized that react with related forms are now recognized. Plasma fibronectin is a solu bullous pemphigoid antibodies. Epidermal pemphigoid antigen ble component of blood (cold-insoluble globulin) while cellular isolated from skin consists of two components: one has a mo (cell smface) fibronectin is insoluble and distributed in fibrillar lecular weight of 20,000 daltons and the other a molecular ru-rays or aggregates loosely bound to cell surfaces and connec weight of 9,200 daltons [39]. Pemphigoid antigen has also been tive tissue matrices. Both forms are similar although not iden isolated from the urine [ 40]. Urinary pemphigoid antigen occurs tical in composition and consist of dimers of 2 subunit polypep predominately as a monomeric form (18,000 daltons), but po tides (200-250 K each) connected near one end by disulfide lymeric forms have also been noted. As might be expected, bonds. Unusual oligosacchrides chains, approximately 4-6 per immunologic identity of pemphigoid antigens derived from polypeptide, consist of a N-acetylglucosamine a nd mannose epidermis, esophagus, saliva and urine was demonstrated using core linked to asparagine residues in the polypeptide subuni t. rabbit anti-epidermal pemphigoid antigen serum. Recently, a These oligosacchride side chains account for a s ignificant por high molecular weight bullous pemphigoid antigen (approxi tion of the biologic activity of these molecules. Cell smface mately 220,000 daltons) was isolated from cultured epidermal fibronectin may also exist in lru·ger multimeric forms. Although cells [ 41]. The relationship of these antigens particularly the fibronectin cannot be considered a unique basement membrane high and the low molecular weight materials is presently un glycoprotein, it is nevertheless a component in a variety of known. Bullous pemphigoid antigen is a product of epidermal basement membranes including skin. Many interesting biologic cells and forms a lamellar layer of BP antigen under basal cells activities have been found for fibronectins in vitro. These even in the absence of basal lamina [ 42,43]. include cell-cell aggregation, cell-substratrum adhesiveness and Laminin: Laminin is a major noncollagenous basement mem specific binding to macromolecul e including collagen, particu brane glycoprotein isolated originally by neutral buffer extrac larly type III collagen. The role of fibronectins in vivo remains tion (soluble laminin) of mouse basement membrane producing to be clarified. Recently, a possible function in wound healing tumor matrix (EHS sarcoma) [44]. Soluble laminin is a large has been suggested. (800-1,000 K) asymmetrical molecule consisting of several sub Composition of the epidermal-dermal junction: Until re units (220 K and 440 .K) that are connected to each other by cently, virtually nothing was known of the biochemical com disulfide bonds. The disulfide bonds are largely localized to 2 position of the structural components of the epidermal-dermal domains that can be separated by pepsin cleavage (Pl and P2) junction. Attempts to isolate junction components has proven and contain 85% and 10% of the bonds respectively. An insoluble difficult owing to the limited accessibility of the epidermal form of laminin. has also been identified [ 46]. Some evidence dermal junction and the relatively small content and antici also suggests that other proteins related to laminin may be pated low yield of these materials. Several recent studies [54- present in basement membranes [ 45,46]. Highly specific anti 56) have reported limited success in this direction but have not bodies produced to soluble laminin have shown that laminin is yet lead to biochemical characterization of the components. immunologically distinct from all other basement membrane Using an indirect approach considerable success has been antigens. Immunofluorescence studies using these antibodies achieved in which highly pmified antibodies to basement mem have demonstrated that laminin or closely related antigens are brane components derived from other sources are used as widely distributed in basement membranes in virtually all tis probes to analyze the composition of the epidermal-dermal sues and species tested [ 4 7-49]. Both epithelial and endothelial junction. By this a pproach, it is possible to localize the isolated cells in culture produce laminin subunits (220 K and 440 K) but basement membrane collagenous and noncollagenous compo mesenchymal cells do not [ 49]. nents to the epidermal-dermal junction utilizing immunofluo Heparan sulfate-containing proteoglycan: Heparan sulfate rescence and ultrastructural immunoelectron microscopic pro contain ing proteoglycan is a large proteoglycan with a molecu cedures. A summary of these results is shown in the Table. lar weight of approximately 750,000 daltons [50]. This material Antibodies to type IV collagen (derived fTom mmine tumor) was also first isolated from EHS sarcoma. Heparan sulfate localized type IV collagen to the basement membrane zone of contain ing proteoglycan contains approximately equal amounts human skin by immunofluorescence [57]. Staining was noted of protein and covalently linked heparan sulfate. Purified anti around skin appendages and vessels as well. By immunoelectron body to heparan sulfate proteoglycan was used to locate this microscopy, antibodies to type IV collagen localize to the basal material in a variety of tissues where it was found to be a lamina only [57,58]. The lamina Iucida and anchoring fibrils, ubiquitous component of basement membranes. dermal microfibril bundles and collagen fibers in the sub-basal Amyloid P-component: Amyloid P-component is a glycopro lamina fibrous area were nonreactive. tein . found in all forms of amyloid and presumably is derived Confusion exists regarding the presence of type V (AB) from serum amyloid P-component, a normal plasma constitu collagen at the epidermal-dermal junction. Stenn, Madri, and ent. The presence of amyloid P-component in normal, nonam Roll [59] reported the localization of antibodies to AB collagen yloid containing tissue 2 was first detected by Schneider and Loos in mouse epidermal-dermal junction. However, Gay et al [60] [51] who observed that anti-amyloid-P component antibody could not demonstrate A and B chains or type V collagen in stained vascular and perivascular structures and suggested a human epidermal-dermal junction utilizing antibodies specific basement membrane localization. This was confirmed by iso for these components. An explanation for these discrepancies is lation of an antigen from glomerular and other vascular base not presently available. ment membranes a nd· demonstration that it'was immunochem Antibodies specific for 7S collagen have demonstrated the ically indistinguishabl e from serum amyloid P [52]. It has not presence of 7S collagen in epidermal-dermal junction by im as yet been proven that this antigen is biochemically identical munofluorescence [38). Immunoelectron microscopy has not with amyloid P component . No immunochemical cross-reactiv been reported as yet to localize 78 collagen to a specific junc ity was detected between this material and other collagenous tional structure. or non-collagenous basement membrane components. These Several noncollagenous glycoproteins have a lso been shown studies suggest that amyloid -P component may be a normal to be present in skin. Antibodies s pecific for laminin derived matrix glycoprotein of some basement membranes. from murine tumor localized to the lamina Iucida on immuno Fibronectin: Fibronectins are a family of high molecula1· electron microscopic studies in human skin. Bullous pemphi weight glycoproteins that a1·e widely distributed in most tissues, goid antigen has also been located in the lamina Iucida [57). particu-larly on the smface of cells and in connective tissue Some studies have found bullous pemphigoid antigen predom matrices. Fibronectins are also found in the plasma and as a inately on the basal cell surface [61). Bullous pemphigoid component of basement membranes . The fibronectins are antigen can also be found on dissociated basal cells after trypsin known by a vru-iety of other names owing largely to theu· vru·ied separation as a fluorescent ru·ea at one pole of the cell indicating somces and interesting biologic activities [53]. Two closely its previous basal localization. Heparin sulfate-containing pro- Jan. 1982 BIOCHEMICAL COMPOSITION OF THE EPIDERMAL-DERMAL JUNCTION 5 teoglycan has been localized to the basement membrane zone (PAM 212) in low concentrations. The ultrastructural localiza of skin using an antibody specific for this material (50]. Immu tion of laminin as well as bullous pemphigoid antigen and noelectron microscopy has not as yet been carried out to fibronectin places these materials in a strategic localization determine its ultrastructural localization. Its distribution in between b asal l amina (type IV collagen) and epidermal cell to other tissues, however, suggests localization in the lamina Iu serve this attachment function. cida. Fibronectin has been demonstrated at the basement mem Hepru·an sulfate proteoglycan and possibly other protein brane zone of skin by immunofluorescent techniques [62-64]. It associated glycosaminoglycans may function in regulating the is also present in other areas of skin including the papillary and permeability of the basement membrane. In kidney, the re reticular dermis. By immunoelectron miCJ·oscopy, fibronectin is moval of heparan sulfate with heparinase significantly a lters localized ultrastructurally to the lamina Iucida, particularly pe1·meability characteristics of membrane [72]. near the basal surface, and in the area below the basal lamina Laminin and type IV collagen may also play an important but not s pecifically associated with sub-basal fibrous structmes role in morphogenesis. Laminin is known to be present from [63]. Basal lamina showed no reaction indicating that fibronec very eru·ly stages in developing t issues [73]. In several tissues it tin is not a component of basal lamina. has been suggested that the selective deposition oflaminin and/ In summary, these data indicate a heterogeneous composition or type IV collagen m ay be a facilitating mechanism in mor of the epidermal-dermal junction composed of both collagenous phogenesis [74]. (type IV, 7S and perhaps type V collagens) and noncollagenous glycoproteins and proteoglycans (laminin, bullous pemphigoid REFERENCES antigen, heparin sulfate containing glycoprotein and fibronec 1. Briggaman R, Wheeler CE Jr.: The e pidermal-dermal junction. J Invest Dermatol 65:71, 1975 tin). 2. Daroczy J , Feldman J , Kira ly K: Human epidermal basal la mina: Its structure, connections a nd functions. Front Matrix B10 l 7:208, Functional aspects of basement membrane components 1979 3. 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