Junctional basement membrane anomalies of skin and mucosa in lipoid proteinosis (hyalinosis cutis et mucosae)

Journal of Dermatological Science (2007) 45, 175—185

www.intl.elsevierhealth.com/journals/jods

Junctional basement membrane anomalies of skin and mucosa in lipoid proteinosis (hyalinosis cutis et mucosae) Nicolae Mirancea a,d,1, Ingrid Hausser b,1, Dieter Metze c, Hans-Ju ¨rgen Stark a, Petra Boukamp a, Dirk Breitkreutz a,* a

German Cancer Research Center, Division ‘Genetics of Skin Cancer’, Heidelberg, Germany Dermatological Department of the University of Heidelberg, Germany c Dermatological Department of the University of Muenster, Germany d Romanian Academy of Sciences, Bucharest, Romania b

Received 21 July 2006; received in revised form 4 November 2006; accepted 16 November 2006

KEYWORDS Lipoid proteinosis; Skin and oral mucosa; Basement membrane; Dermo-epidermal junction; Immunoelectron microscopy

Summary Background: Excessive basement membrane (BM) deposition in skin and mucosa is characteristic for lipoid proteinosis (LP; hyalinosis cutis et mucosae), an inherited disease caused by extracellular matrix protein 1 (ECM1) mutations. According to ultrastructure there are striking differences between junctional and microvascular BM. Objective: Distinct analysis of the junctional zone in epidermis and oral mucosa, contrasting concentric BM arrays in the microvasculature; evaluation of impact on epithelial histogenesis and differentiation, and specifically on adhesion structures to BM (hemidesmosomes). Methods: LP-epithelia were analyzed for alterations in differentiation, BM composition and texture, and hemidesmosomal components by indirect immunofluorescence (IIF), electron microscopy (EM), and immunoelectron microscopy (ImEM). Results: Most striking was the irregular deposition of collagen IV and VII, BM-laminin, and laminin-5 at the junctional zone, accompanied by lamellate or punctuated structures below BM (IIF), whereas integrin a6b4 and bullous pemphigoid antigen1 and -2 (BPAG-1/-2) were regularly aligned. Also integrins a2b1 and a3b1 remained restricted to the epidermal basal layer, while the tissue-specific differentiation

Abbreviations: BM, basement membrane; BPAG, bullous pemphigoid antigen; ECM1, extracellular matrix protein 1; EHS, Engelbreth—Holm—Swarm (transplantable BM producing tumor); EM, electron microscopy; IIF, indirect immunofluorescence; ImEM, immunoelectron microscopy; LP, lipoid proteinosis * Corresponding author at: DKFZ Heidelberg, Div. A110 (A080), P.O.B. 101949, D-69009 Heidelberg, Germany. Tel.: +49 6221 424511; fax: +49 6221 424551. E-mail address: [email protected] (D. Breitkreutz). 1 Both authors contributed equally well to this work. 0923-1811/$30.00 # 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2006.11.010

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N. Mirancea et al. markers keratin K1/10 (mucosa, additionally K4/13) appeared delayed indicating mild hyperplasia, further confirmed by focal K6/16 expression. Ultrastructure (EM) disclosed abundance of extended basal cell protrusions and junctional aberrations like exfoliating excessive BM material. Hemidesmosomes were complete, but ImEM indicated weakened interactions between their components (BPAG-1, -2, and HD1). Confirming IIF, collagen IV and VII, and laminin-5 appeared extensively scattered, the latter two probably remaining associated. Conclusions: Subtle defects in anchorage assembly, spanning the entire BM zone, apparently compromise epithelial-matrix adhesion, which may provoke (mechanical stress-induced) erroneous BM repair. # 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction Lipoid proteinosis (LP; hyalinosis cutis et mucosae; OMIM 247100) is an autosomal recessive condition displaying multiple systemic manifestations. Mainly affected are skin, mucosa of the upper aerodigestive tract, and the local microvasculature. Typical clinical symptoms are a hoarse voice, itching bullous skin eruptions healing with atrophic scars, and generalized yellowish infiltrations of skin and mucosae (reviewed by [1,2]). Main characteristics of LP-histology are hyaline deposits within the interstitial connective tissue and abnormal small blood vessels with extensively thickened vessel walls. Common ultrastructural features are irregular multiplications of basement membrane (BM) structures at the dermo-epidermal junction and onion shell-like BM arrays around microvessels ([3,4] and references therein). Accordingly, excessive deposition of BM constituents, like type IV collagen, BM-laminin [5] and other major BM molecules [4], has been found by indirect immunofluorescence (IIF). The tissuespecific defects in LP-tissue may relate to differences in composition and cellular source of BM molecules. Whereas vascular endothelial cells produce their entire BM complement, for the junctional BM keratinocytes synthesize all major components except nidogen, which is provided by the neighboring fibroblasts. Crucial for firm anchorage in skin are type VII collagen and epithelial-specific laminin-5, both interconnected within the BM zone [6,7]. The markedly enhanced type IV collagen synthesis in fibroblast cultures, derived from LP-skin [8], could imply a particular role of this cell type for the junctional aberrations in the disease. Mutations in the extracellular matrix protein 1 (ECM1) are the common cause of LP [2,9,10]. While the type of mutation, leading to partial or complete ECM1 loss-of-function (depending on the affected splice variants), largely determines the clinical symptoms; there is considerable variation among patients with identical mutations [9,10]. Thus, the individual microenvironment modulates the

degree of disturbance in the balance of BM synthesis and turnover, presumably involving interactions with other matrix components. In addition, the differences between BM anomalies of skin or mucous epithelia and on the other hand the microvasculature raise further questions for the tissue-specific function of ECM1 in regulatory events and molecular interactions. In our biopsy material from LP-patients the overall epithelial structure (skin, mucosa) appeared largely normal, besides some lesions in exposed areas and general mild hyperplasia. The existence of multiple BM arrays, seen by EM, was corroborated by large deposits of nidogen, BM-laminin, type IV collagen, and perlecan (IIF, in part immuno-EM), consisting with previous reports on LP-skin [5] and its microvasculature especially [4]. Herein we have focused on the correlation of BM alterations with the fate of junctional complexes, not explicitly addressed in earlier studies. While in our specimens molecular arrays responsible for dermal anchorage of BM (type VII collagen, laminin-5) were more visibly affected than the intracellular hemidesmosomal constituents, both together may alter adhesive properties and thus, provoke erroneous, unscheduled BM repair processes.

2. Materials and methods 2.1. Biopsy material Biopsies from skin or small flaps of oral mucosa removed at tooth extraction were taken with LPpatients’ (parents’) consent on occasion of medical examination. As common clinical symptoms, patients were suffering from pronounced hoarseness, infiltrations within the oral cavity and the larynx, and skin lesions at exposed areas like elbow, forearm, and back of hands. Patient 1 was a 28-year-old Turkish female, one of two affected siblings [3]. Biopsy material from axilla and face (near the eyebrow), and a flap of oral mucosa (recovering from acute parotitis) were

Junctional basement membrane in lipoid proteinosis examined by light microscopy, indirect immunofluorescence (IIF), and transmission electron microscopy (EM). From patient 2, a Greek boy, a tissue sample from forearm was taken at the age of seven and prepared for light microscopy and EM (described in [11]). Patient 3, a Turkish girl, was clinically examined at the age of 5 and 10. Biopsies were taken from forearm, elbow, and oral mucosa (tissue removed during dental treatment) and processed for IIF, EM, and immunoelectron microscopy (ImEM).

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2.3. Electron microscopy (EM) Fresh tissue samples were fixed in 2.5% glutaraldehyde (in cacodylate buffer pH 7.2), followed by 2% OsO4, stained en bloc with uranyl acetate, and embedded in Epon 812-equivalent (Serva, Bioproducts, Heidelberg, Germany); ultrathin sections were counter-stained with uranyl acetate and lead citrate [4,18].

2.4. Immunoelectron microscopy (ImEM) 2.2. Histology and indirect immunofluorescence (IIF) For initial histological examination tissue samples were fixed and processed as for EM (described below); semithin sections of these resin-embedded specimens were stained with methylene blue. Indirect immunofluorescence (IIF) was performed on fresh or pre-fixed material (2% formaldehyde and 0.05% glutaraldehyde; used for ImEM) as in previous studies [4,12]. In brief, cryostat sections (8 mm setting) were fixed with methanol-acetone (1 + 1) for 5 min at 20 8C, rehydrated, incubated in blocking buffer (1% bovine albumin in PBS), and then with primary antibodies overnight at 4 8C. After washes with blocking buffer secondary antibodies were applied for 30 min at RT. The following primary antibodies, adjusted with blocking buffer to final working dilutions, were used (described in [12—14]). Rabbit antibodies against bullous pemphigoid antigen 1 (BPAG-1, BP230) were a generous gift from J. Stanley (Bethesda, Maryland, USA), BPAG-2 (BP180/type XVII collagen; juxtaposed extracellular domain [15]) from L. Bruckner-Tuderman (Freiburg, Germany), laminin5 from P. Marinkovich (Stanford, California, USA), and the respective laminin g2 chain from G. Meneguzzi (Nice, France). Monoclonal mouse antibodies against type VII collagen (C-terminus) were donated by I. Leigh (London, UK); integrin a2, a3 and b1 chains by E. Klein (E. Bro ¨rzburg, Germany), rat mono¨cker, Wu clonal antibodies against integrin a6 (G0H3) by A. Sonnenberg (Amsterdam, The Netherlands), and integrin b4 (439/9B; extracellular domain) by R. Falcioni (CRS, Rome, Italy). Goat and rabbit antibodies against BM-laminin (isolated from EHS-tumor [16]) and type IV collagen were donated by J.-M. Foidart (Liege, Belgium) or purchased from Progen (Heidelberg, Germany), rabbit anti-nidogen (nidogen-1) donated by R. Nischt and R. Timpl (Cologne and Martinsried, Germany; [17]), and rabbit antiloricrin by D. Hohl (Lausanne, Switzerland). Secondary antibodies were purchased from Biotrend (Cologne, Germany), Dianova (Hamburg, Germany), and Molecular Probes (Leiden, The Netherlands; MobiTec, Germany).

As described previously, for immunoelectron microscopy (ImEM) a mild fixation procedure was applied (2% formaldehyde, 0.05% glutaraldehyde in PBS; [4,12]), testing the reactivity of the individual primary antibodies beforehand by IIF. After 1 h the reaction was stopped with 50 mM NH4Cl, specimens were incubated in 3.4% sucrose (30 min), dehydrated and embedded in Unicryl resin (British BioCell, Cardiff, UK). Ultrathin sections collected on nickel grids were incubated with primary antibodies overnight and, after thorough washing, with goldconjugated secondary antibodies (<1, 5, 10 or 15 nm; Aurion/BioTrend, Cologne, Germany) at RT for 2—5 h. When applying <1 nm (ultra-small) gold conjugates, the reaction was silver enhanced (SE) following the manufacturers’ protocol (Aurion); counter-staining was like for conventional EM.

3. Results 3.1. Clinical picture and histopathology (light microscopy) Comparable to the other patients, the affected skin of patient 2 (Fig. 1a) revealed scarring (elbow) and small reddish papules (forearm, and back of the hand). In addition, physical examination had disclosed long-standing varicella-like atrophic scars and hyperpigmentation on face and limbs. Histological sections of LP-skin (Fig. 1b) and oral mucosa (shown elsewhere [4]) generally revealed a mild epithelial hyperplasia and serrated contours at the cell—matrix junction. Numerous microvessels with prominent thickened walls (arrows; detailed description in [4]) and stromal cells were located in close vicinity to LP-epithelia.

3.2. Tissue markers and components in the BM-zone (indirect immunofluorescence) The patterns of epidermal markers in LP-skin, seen by indirect immunofluorescence (Figs. 2 and 3; IIF;

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Fig. 1 Clinical picture (a) and histology (b) of LP-skin. Around the elbow a verrucous hyperkeratotic area is seen, on forearm and hand hyperpigmentation and atrophic scars (a). The semithin section of affected skin (elbow) reveals a ‘serrated’ epidermal outline at the junctional zone (b, inset), abundant small blood vessels with thickened walls (arrows), and hyaline deposits (b). E, epidermis; scale bars, 100 mm.

patient 3) confirmed the slightly hyperplastic phenotype. Thus, onset of keratin K1 and K10 expression was irregularly shifted towards upper suprabasal layers (Fig. 2a), accompanied by expression of the ‘hyperproliferative’ keratins K6 and K16 (not shown; [14]). In both locations, epidermis and mucosa, overall tissue architecture and other epithelial markers did not reveal striking anomalies. The cell—cell interfaces were regularly outlined by desmogleins, desomoplakins, and plakoglobins delineating desmosomal attachment sites like in healthy tissues; oral mucosa additionally contained keratins K4 and K13 (not shown), specific for stratified internal epithelia ([19] and references therein). The BM zone in LP-skin revealed a regular lining by the integral hemidesmosomal proteins BPAG1 (BP230; Fig. 2a) and BPAG2 (BP180/type XVII collagen; Fig. 2b), the latter displaying also filigree surface decoration of basal cells. Co-staining for nidogen confirmed discrete BM alignment of the bulk-part of BPAG2, while emphasizing the thickening of vessel walls (Fig. 2b; underneath epidermis). The distribution of integrins a2b1 and a3b1 (Fig. 2c, a3 chain) resembled that of BPAG2, though being more pronounced pericellularly, while integrin a6b4 was virtually restricted to the BM zone, broadened in some areas apparently for tilting of BM (Fig. 2d0 and d00 , arrows; a6 chain). Laminin-5 displayed virtually complete colocalization with integrins in the BM zone (Fig. 2c, d and d0 ), but locally some laminin-5 shedding became apparent

Fig. 2 Alterations of epidermal differentiation and BM zone of LP-skin shown by indirect immunofluorescence (IIF). Mild epidermal hyperplasia becomes apparent by irregular shifts in the onset of keratin K1/K10 expression from the first to upper suprabasal layers (a); regular lining of the BM zone by the hemidesmosomal protein BPAG1 (BP230; red) is maintained (a). Intense BM labeling is also seen for BPAG2 (BP180; green), showing in addition filigree surface decoration of basal cells (b); the colocalized BM protein nidogen (red, yellow overlap) demonstrates in addition thickening of vessel walls (red; arrows). The pattern of integrin chain a3 (c; red) resembling BPAG2 (though more pronounced pericellularly) coincides in the BM zone (yellow) with laminin-5 (c; green) which also shows some shedding (insets). The integrin a6 chain (red) is restricted to the BM zone, sometimes broadened for tilting of BM (d00 ; arrows), largely matching the linear laminin-5 pattern (d0 ); laminin-5 shedding (d, green) is emphasized by higher magnification. Blue nuclear stain in (a, d0 ); E, epidermis; scale bars, 100 mm.

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Fig. 3 Altered distribution of type IV (a, epidermis) and type VII collagen (b, oral mucosa) in LP. Extended lamellar structures are observed for type IV collagen (a; green) which also demarcates thickened vessel walls, confirmed by counterstaining of vascular endothelia by CD31/PECAM (a; red). Apart from regular linear BM lining, type VII collagen (b, red) shows in some areas distinct punctuate patterns below the BM zone (inset); epithelium is stained by a pan-keratin antibody. E, epidermis or epithelium; scale bars, 100 mm.

(Fig. 2c, insets), better visible at higher magnification (Fig. 2d and d0 ). Turning to the major structural BM component type IV collagen, lamellar shedding was even more pronounced extending far into the dermis underneath (Fig. 3a). Further, as shown for nidogen above (Fig. 2a), this was sharply demarcating thickened vascular structures, counterstained by the endothelial cell surface marker CD31/PECAM (Fig. 3a). A comparable picture was obtained for BM-laminin (g1-forms, mainly laminin-10), which has been demonstrated recently [4]. Staining for type VII collagen, responsible for BM anchorage, disclosed a continuous regular BM lining (Fig. 3b; oral mucosa) but in some areas additionally distinct punctuate patterns in the adjacent connective tissue (Fig. 3b and inset). This was found likewise in mucosa (Fig. 3b; epithelial pan-keratin counter-staining) and in skin (not shown).

3.3. Ultrastructure of the dermoepidermal junction (electron microscopy) At the ultrastructural level, very prominent and typical for LP were numerous basal cell protrusions deforming the junctional zone (Fig. 4a, skin of patient 1 and 4b, patient 3), though in general

Fig. 4 Ultrastructure of cutaneous BM zone of patient 1 (a) and 3 (b). BM lining is largely continuous except at the small cell aspect rich in melanosomes (a, arrowhead). Multiple displaced BM structures (thick arrows) are intermingled with fibrils of interstitial collagen, seen in cross and tangential section (a, insets). Hemidesmosomes and anchoring filaments have a regular appearance, while anchoring fibrils (b, thin arrows) are spread to a large part, often associated with multiple basal laminae and collagen fibrils (a and b, insets). Scale bars: a, 1000 nm and b, 500 nm.

the BM was continuous and hemidesmosomes were well developed, revealing regular connections to the lamina densa by anchoring filaments and below to the fibrillar collagen by anchoring fibrils. On the other hand multiple reduplications of BM were abundant, particularly alongside basal cell projections, and BM structures together with attached anchoring fibrils were exfoliating into the papillary dermis (Fig. 4a and b). But this was by no means matching the vast repetitive BM patterns seen around the adjacent small vessels (documented elsewhere [4]). The lack of lamina densa at the cell process loaded with melanosomes (Fig. 4a) seems to correlate to gaps in BM staining beneath melanocytes observed by others applying IIF and confocal imaging [20].

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Fig. 5 Ultrastructural localization of hemidesmosomal components in healthy (a—c; HS) and LP-skin (d-f; LP) by immuno-electron microscopy (ImEM, immuno-gold staining). In both cases BPAG2 (BP180; external domain) is mostly associated with the sub-basal dense plate of hemidesmosomes (a and d). Labeling for BPAG1 (BP230) matches the intracellular outer dense plaques (b and e) with occasional distortion in LP-samples especially in finger-like cell protrusions (e). In both specimens HD1 is positioned in the area of inner dense plaques (c and f); in most cases detection was improved by applying smaller seized gold-particles (c vs. inset; +SE, see below). In (f) double-label for HD1 (small particles) and type IV collagen is shown; inset in (f), en face view of adhesion plane, presumably better exposed. Gold particles: a, 15 nm; c and e (large area), f (inset), 10 nm; b, c (inset), d, e (right inset), ultra-small (<1 nm) and silver enhancement (SE); SE also in f (large; double label), HD1, 5 nm and CIV, 10 nm; scale bars, 250 nm.

Fig. 6 Ultrastructural localization of matrix proteins of the BM zone in healthy (a—c, HS) and LP-skin (d-f, LP) by ImEM. Type IV collagen (a and d) is arranged in a regular band, fully coinciding with lamina densa in normal skin (a), while in LP (d) also disconnected strands are seen; double label (type IV collagen, 10 nm gold) indicates preferentially regular association of integrin b4 chain (5 nm) with the sub-basal dense plate (left inset; single b4 label lower right). Laminin-5 (b and e) appears in normal skin clustered at hemidesmosomal sites at the upper face of lamina densa (a). Regular spacing of laminin-5 is partly maintained in LP-skin (e), but a considerable part is scattered remote of the BM zone. Staining of type VII collagen (c, f) follows normally the lamina densa (c, HS) like type IV collagen. The type VII pattern is partially conserved in LP (f, inset) besides intensive spreading similar to laminin-5 (further details in Fig. 7). Thick arrows, visible lamina densa; thin arrows, sub-basal dense plate; gold particles: a and b (both, large area), 10 nm; a and b (insets), c and d (large), e and f, ultra-small (<1 nm) and silver enhancement (SE); d (insets), b4, 5 nm and CIV, 10 nm; scale bars: a, b, e, f, 500 nm; c and d, 1000 nm.

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3.4. Molecular displacement of junctional and basement membrane components (immunoelectron microscopy) The comparison of the ultrastructural localization of hemidesmosomal and junctional components in normal (Fig. 5a—c) and LP-skin (Fig. 5d—f; patient 3) by immunoelectron microscopy (ImEM) disclosed only minor distortions. In both cases BPAG2 (BP180; external juxtamembranous domain [15]) was mostly associated with the sub-basal dense plates of hemidesmosomes (Fig. 5a and d) and BPAG1 (BP230) with corresponding outer plaques intracellularly (Fig. 5b and e). But generally clustering of HD-components was slightly less pronounced in LP-patients. Particularly HD1 (plectin), a major constituent of inner plaques [21], was located slightly more remote from the surface in LP (Fig. 5c versus f), apparent by double-labeling with type IV collagen (5f, lower panel). However, for limited access to the epitopes within these plaque structures any interpretation has to be dealt with caution. Contrarily, there were marked discrepancies in the localization of matrix-components of the BM zone between normal (Fig. 6a—c) and LP-skin (Fig. 6d—f). Labeling of type IV collagen (Fig. 6a and d), regularly detectable as distinct band coinciding with a visible lamina densa (6a), disclosed in LP a far less defined BM lining (Fig. 6d). Thus, to a large part the distribution was either diffuse or corresponding to the displaced basal lamina stretches seen by EM (Fig. 4). Double label of type IV with the integrin b4 chain indicated preferential association of b4 (external domain) with sub-basal dense plates also in LP (Fig. 6d, insets; not shown for normal skin) similar to BPAG2, confirming overall basal cell integrity. Antibodies to laminin-5 (Fig. 6b, e) aligned in normal skin on top of lamina densa, facing basal cells and being clustered along anchoring filaments below hemidesmosomes (Fig. 6b). In part this regular spacing was maintained in patients’ skin (cell processes in Fig. 6e, inset), whereas in other areas the laminin-5 label was largely diffused. Comparable patterns or changes were observed for type VII collagen (Fig. 6c versus f). In normal skin staining followed the lamina densa (Fig. 6c), clustered underneath hemidesmosomes at anchoring fibrils attachment sites. While this spatial association was partially preserved in LP, there was similarly intensive spreading (Fig. 6f), which may indicate persisting associations with laminin-5. The variance of structural appearance in the microenvironmental context is emphasized in Fig. 7. In regions with massive bundles of type I collagen fibrils distinct regular configurations are seen (Fig. 7, and insets on the right), while next to it

Fig. 7 Spatial associations of type VII collagen in well structured area of LP-skin. Regular arrays indicating normal interactions with end-points of anchoring filaments within the lamina densa (insets; lower right, frame outside the field) at sites close to massive bundles of fibrillar collagen (seen in cross-section). Adjacent, the displaced clustered arrays of type VII (left inset) seem to correspond to dissociated anchoring fibrils (comparable to Fig. 2). Gold particles, ultra-small (<1 nm) and silver enhancement (SE); scale bar, 1000 nm.

labeled clusters are widely displaced (Fig. 7, left inset) corresponding to the remote complexes of anchoring fibrils and lamina densa strands shown above by EM (Fig. 4). Collectively, the epithelial anchoring system as a whole seemed to be locally compromised in LP-samples.

4. Discussion Irregular deposition and accumulations of basement membrane (BM) represent special features of lipoid proteinosis (LP), affecting skin, mucous epithelia and especially small blood vessels. This could be due to lack of feedback control in synthesis, processing, secretion or assembly but also reduced turnover. The primary cause are mutations in the ECM1 gene, abolishing ECM1 synthesis or leading to partial loss-of-function [1,2,9,10] and accordingly autoantibodies against ECM1 (lichen sclerosus [1,22—24]) or decrease of ECM1 during aging [25] have been reported to cause similar symptoms. To illuminate further facets of ECM1 function, herein we have focused on the disturbed molecular architecture of the junctional zone in LP, including hemidesmosomal adhesion complexes. In the patients of this study the overall epithelial phenotype (skin, mucosa) appeared largely normal, besides general mild hyperplasia as judged by differentiation markers. The excessive BM structures, seen by EM, corresponded to large deposits of BM-laminin (in skin laminin-10), type IV collagen, nidogen, and perlecan (IIF). But in contrast to the uniform concentric arrays of multiple vascular BMs

182 [4], irregularities of the junctional BM zone were far more variable, accompanied by frequent dissociation and displacement of anchoring complexes. At least four major aspects have to be taken into account to explain the peculiar histopathology: (i) tissue specific composition of BM, (ii) diverse cell sources of these molecules, (iii) related to that, distinct mechanisms of BM assembly, and (iv) in return different responses of the matrix aligned cells. Most obvious, junctional and vascular BM differ in their laminin isoforms. Laminin-10 predominates in cutaneous BM, accompanied by short-armed laminin-5, whereas laminin-8 and 10 are co-expressed in vascular endothelia [26— 28] varying in their interactions with other BM molecules. Most laminins (with a g1-chain; not laminin-5 [26]) bind strongly nidogen-1, prevailing in skin and mucosa [29], whereas in vascular BM nidogen-2 is markedly increased (being in skin dispensable), which binds only weakly to laminin g1 [17]. The four main BM-components are synthesized by fibroblasts or vascular endothelial cells, while epithelial cells do not synthesize nidogens but instead laminin-5 [30]. The complete set of BM molecules made by growing vessels ensures fast BM assembly, promoting vascular stabilization and tight seal. In mice deprived of both nidogens (perinatal lethal), small vessels generally lacked BM structures and were leaky (own data, Nischt et al., in preparation1). While the junctional BM was only mildly affected in these newborn mice, in a skin-organotypic coculture model nidogen was essential for junctional BM formation, which in return was accelerated by supplying either nidogen form [12,31]. Critical functions of the junctional BM are to warrant firm anchorage of epithelia and resistance to robust mechanical forces [7,28]. According to wound and regeneration models, this requires stepwise assembly, which will provide an optimal fit of all involved components. Crucial for this task in skin are integrins [32,33] as well as the matrix molecules laminin-5 and type VII collagen (the latter contributed by epithelial cells and fibroblasts), both interacting with each other as integral parts of the compound anchoring complex [6,34,35]. Thus, besides the pivotal role of soluble factor mediated cross-talk, the BM per se represents a common working platform for both cell types [30]. In LP a regional dissociation of these anchoring structures from epithelial sites was seen already by IIF, but more clearly by EM. This seems to be a plausible 1

Baranowsky A, Mirancea N, Mokkapati S, Smyth N, Hausser I, Breitkreutz D, Nischt R (2007). Distinct junctional and vascular basement membrane defects in nidogen null mice.

N. Mirancea et al. reason for the abundance of basal cell protrusions, reflecting local retraction of cells at weakened attachment sites. Compensatory BM-repair then could give rise to the tangling multi-lamellate structures and loosely associated anchoring fibrils. Being further substantiated by ImEM, the patterns imply the persistence of detached laminin-5/type VII collagen complexes [6,34,35] after dissociation from regular sites. How far this affects the functional integrity of internal hemidesmosomal structures has to be interpret with caution. While localization of the transmembrane components BPAG2/type XVII collagen [15] and a6b4 [32,36] appeared normal in LP, the external epitopes by and large coincide with sub-basal dense plates, detection of internal plaque components was severely hampered by steric hindrance. In most cases this afforded more sensitive detection using smaller sized gold-particles (with silver enhancement) and compromising structural conservation (mild fixation procedure). Weakening of internal linkages, involving also HD1/ plectin [21,36], to keratin filaments would be compatible with ‘free-floating’ desmosomes in LP [37], also common in edema and spongiosis (e.g. Netherton syndrome). Furthermore it would explain blistering in bullous forms of lichen sclerosus, an acquired skin disease with comparable ultrastructural disturbances where ECM1 function is affected by autoantibodies [22—24]. Finally, it remains obscure to what extent type IV collagen is interacting in LP, being destabilized in junctional BM, but forming distinct concentric arrays around vessels in line with the multiple BM lamellae [4]. Accordingly, the ultrastructural distribution of laminin-10 (similarly enhanced according to IIF [4]) in comparison to type IV would be of greatest interest; both structural core components of BM form principally independent networks [28]. An experimental proof for that last point provided the aforementioned skin-organotypic model, where complete blockade of laminin assembly or BM formation did not suppress alignment of a type IV collagen scaffold, though it was less structured [12]. Major questions to ask now are, at which stages and how ECM1 is involved in BM formation and proper maintenance? Does ECM1 engage in persisting molecular interactions or play a regulatory role, functioning as adapter, transporter or shuttling device? Structural analysis of ECM1 had revealed typical domains for protein-protein binding, implying interplay with various regulatory factors or receptors, known for transport proteins like albumin [38]. Crucial interaction partners in this context could be the BM components perlecan [39,40] and fibulin-1 [41], both strongly binding to ECM1 depending on the particular splice variant. Of the

Junctional basement membrane in lipoid proteinosis known forms ECM1a, b, and c, only ECM1a is abundant, present also in skin where it decorates most strongly epidermal basal cells [38]. In LP most mutations occur in exon 7, coding for the second repeat in ECM1a with the fibulin-1 recognition site. The result is a frame shift leading to premature termination, which also deletes the perlecan binding site [2,10]. This may be especially critical, since both perlecan and fibulin-1 interact with most other BM molecules and have also regulatory functions, binding or releasing growth factors and cooperating with corresponding receptors. Upregulation of ECM1 has been found in tumors [42,43], where it probably promotes angiogenesis [23,40,42]. Furthermore, elevated levels of the binding partners perlecan and fibulin-1 were detected in cancer [43—45] and herein the high affinity of fibulin-1 to nidogen [46] could be crucial for vascular BM remodeling. Interestingly in this context, perlecan seems to play a rather functional than structural role in BM, exemplified by its strong anti-apoptotic effect in a skinorganotypic coculture model ([47]; and own unpublished data). Molecular aspects regarding the microvasculature had been discussed already in more depth elsewhere [4]. Lastly, also minor BM associated collagens could be involved such as type XV or XVIII, which may engage in control of molecular BM dimensions [48]. Those non-fibrillar collagens (multiplexins) have gained much attention in recent years for the anti-angiogenic properties of their C-terminal fragments restin and endostatin, respectively [49,50]. Still there remains the question, is there any connection to BM turnover? Highaffinity binding of the matrix metalloproteinase MMP9 to ECM1 has been reported very recently [51]. However, protease activity was considerably inhibited, implying more intriguing mechanisms maybe involving local concentration and thus spatially controlled release of active factors and bioreactive peptides or processing of other proteases [52]. To conclude, the phenotype resulting from mutations of ECM1 is clearly different from other genodermatoses. This assigns ECM1 a specific role in BM formation or functional maintenance. Among others, our studies demonstrate that quite distinct mechanisms are acting in building up the junctional BM zone of stratified epithelia versus microvascular BM with extremely thickened vessel walls. In the epithelial BM zone, more critically affected are ‘stable’ anchoring structures, mostly evident at the ultrastructural level. Although effects on epithelial adhesion appear rather mild compared to classical inherited bullous diseases, this may cause chronic epithelial irritations and bullae especially in childhood. Later infiltrations and veruccous

183 epidermal thickening will then be accompanied by varying reduplications of BM and detached anchoring complexes hampering further balanced assembly. Site-directed mutagenesis in transgenic animals in combination with skin-organotypic models [14] and gene silencing should shed further light on cellor tissue-specific functions of the still enigmatic ECM1. Prerequisite for that are certainly mutational analyses of the involved cases, which are now in progress. So far a new homozygous ECM1 mutation has been discovered in one of these patients (collaboration with H.C. Hennies, personal communication; Cologne Center for Genomics, Cologne, Germany). According to those data, presumably this gives rise to a defective protein, which still has to be confirmed biochemically.

Acknowledgments We like to thank Anke Wollschla ¨ger, Regina Beck, and Renate Ayubi for skillful technical assistance as well as the photo department at the DKFZ for continuous support. This investigation was in part supported by the Deutsche Forschungsgemeinschaft (D.B.) and an industrial grant (N.M.; Aventis Pharma Deutschland/Aventis-Sanofi).

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