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First published online May 24, 2004
doi: 10.1242/10.1242/jcs.01127

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Inhibition of basement membrane formation by a nidogen-binding laminin 1-chain fragment in human skin-organotypic cocultures

¹ German Cancer Research Center, Division A080, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany
² Romanian Academy of Sciences, Spaiul Independentei 296, 77703, Bucharest, Romania
³ Aventis Pharma Deutschland GmbH, Industriepark Hoechst H811/815, 65926, Frankfurt am Main, Germany

View larger version (98K): [in a new window]   Fig. 1. Maintenance of morphology and differentiation in organotypic 3D-cocultures in the presence of laminin 1-fragment (Lf). Histology of epithelia at 14 (A,B) and 19 days (C,D), grown without (A,C) and with Lf (B,D) reveals comparable stratification including defined granular (arrows) and cornified layers (SC), but an increased tendency for splitting at the tissue interface with Lf (D; arrowhead). (E-H), Analysis of Lf-treated tissue by indirect immunofluorescence (IIF) indicates a largely regular expression of the early epidermal differentiation markers keratin K1 and K10 (E) as well as the intermediate and late markers K2e (F), epidermal transglutaminase (G), and loricrin (H). Scale bars: 50 µm.

View larger version (86K): [in a new window]   Fig. 2. Influence of Lf on deposition of basement membrane (BM) components shown by indirect immunofluorescence (IIF) at days 10 (A-F), 14 (G-K) and 19 (L,M). Compared to controls (left column) treated epithelia (right column) have lost or dramatically reduced nidogen (A,B), BM laminin (C,D and G,H) and perlecan (L,M). For laminin detection either antibodies against the laminin 1 chain (C,D) or laminin from EHS tumours (G,H) were employed. By contrast, type IV collagen (E,F and I,K) and laminin-5 (see Figs 3, 4) are only mildly affected by the treatment. Scale bars: 50 µm.

View larger version (86K): [in a new window]   Fig. 8. Protein analysis by western blotting of the separated epithelial and `dermal' compartments (collagen matrix with resident fibroblasts). Extracts were prepared from 3D-cocultures (–/+ Lf treatment as indicated) grown for 12 (left panel) or 14 (right panel) days at the air-liquid interface. Reactions with antibodies against the laminin 5 (L5) and 1 chain (L1), and against nidogen (ND) are shown, the lower part of third blot (*) demonstrates comparable sample loading. The size of the bands of presumptive BM components is indicated in kDa.

View larger version (59K): [in a new window]   Fig. 3. Dissociation of assembled BM components by delayed Lf treatment (starting at day 10) and analysed at day 14. Nidogen (A) and BM laminin (C; detection of 1 chain) are almost absent after this short treatment, whereas the linear deposition of both type IV collagen (B; but some diffuse dislocation into gel matrix) and laminin-5 (D; 2 chain) persists. Scale bars: 50 µm.

View larger version (95K): [in a new window]   Fig. 4. Reversibility of the Lf-effect on the deposition of BM components. Upon withdrawal of Lf at day 10 the deposition of nidogen (A) and BM laminin (C; 1 chain) is largely or partially recovered, whereas continuous treatment (B,D) reveals, at best, only marginal binding to the surface of basal cells. By contrast, there is only little change, at most a moderate reduction in staining for laminin-5 (E,F; 2 chain) and type IV collagen (G,H) between these pairs (control/treated). All shown at day 19. Scale bars: 50 µm.

View larger version (166K): [in a new window]   Fig. 5. Loss of ultrastructural elements of the BM zone caused by Lf. At 14 days untreated 3D-cocultures (A,C) reveal a mostly continuous BM (arrows indicate the lamina densa) and regularly structured hemidesmosomes (arrowheads in C; detailed view) linked to prominent keratin filament bundles (kf). Treatment with Lf not only prevents assembly of BM, but also of hemidesmosomes (B,D) and the association of keratin filaments (kf). Basal cells seem to attach frequently to type I collagen fibrils which can be seen in cross and longitudinal section (D; some contacts are indicated by short arrows). e, epithelium; m, matrix. Scale bars: 250 (A,B) and 100 nm (C,D).

View larger version (79K): [in a new window]   Fig. 6. Persistence of adhesion molecules in 3D-cocultures at 14 (A-D), 19 (E,F) and 21 days (G-K). The patterns of controls (left column) generally undergo only minor changes in the presence of Lf (right column) as demonstrated for the integrin chains 2 (A,B), 3 (C,D), v (E,F), 6 (G,H), and bullous pemphigoid antigen 2 (I,K; BPAG2/BP180). In treated samples, 2 (B) is slightly stronger in basal cells, while the distribution of 3 (D) appears slightly more pericellular and v (F) is less polarised. Further the prominent co-localisation of 6 and BPAG2 in controls (G,I) is not maintained upon treatment with Lf (H,K), indicating extensive shedding of 6 (H) and a mostly pericellular label of basal cells with BPAG2 (K). Arrowheads in D,F,K indicate the partially labelled surface of the collagen matrix in split areas. Scale bars: 50 µm.

View larger version (150K): [in a new window]   Fig. 7. Localisation of BM components and adhesion molecules in control (left column) and Lf-treated epithelia (right column) at 14 days. The control shows a band of denser gold labelling for type IV collagen (A) which becomes extensively dispersed and dislocated in the presence of Lf (B; larger area, about two-fold lower magnification). Similar effects are seen for laminin-5 (C, D; 10 nm gold) which by double labelling also reveals a looser association of the integrin ß4 chain than in controls (C versus D; 5 nm gold). In addition, and in contrast to regular clusters of bullous pemphigoid antigen 1 (E; BPAG1/BP230; 10 nm), only a few single gold particles are seen in Lf-treated specimens (F; outside the field, vast areas without any label). Consequently keratin-positive filaments (`pan'-keratin; 10 nm), normally attached to the hemidesmosomes (G), are, in treated samples (H), largely retracted from the basal cell pole, which is partially demarcated by ß4 (5 nm). In A and B 1 nm gold particles were used together with silver enhancement. e, epithelium; m, extracellular matrix. Scale bars: 150 (A,C-H) and 300 nm (B).