Major BM components and cysteine cathepsins B, K, L, S in the epidermis of human skin.

<p>(A) Immunofluorescence labeling of major BM components (type IV collagen, laminin, nid-1 and -2) and cathepsins B, K, L, S. The nuclei of keratinocytes were stained with DAPI. Ep: epidermis; De: dermis, Sc: stratum corneum, Sg: stratum granulosum, Ss: stratum spinosum, Sb: stratum basale. The continuous line indicates the outermost layer of the skin, the dashed line indicates the basal lamina. Bars correspond to 25 µm. (B) Expression of pro- and mature cathepsins B, K, L and S in human skin epidermis. The dark arrow indicates the proform of cathepsin and the light arrow indicated the mature form. Intermediate bands correspond to uncompleted maturation. (C) Samples of conditioned culture medium of human primary keratinocytes were concentrated (x 200), separated (100 µg protein) by SDS-PAGE (15%) under reducing conditions. (D) Transwell BM matrix migration assays (n = 5 separate experiments) by keratinocytes (C: control) treated with E-64 (**, <i>P</i><0.01 when compared to control). (E) Double immunofluorescent labeling of catS and nid-1 in human skin and confocal laser microscopy analysis. Secondary antibodies were Alexa Fluor 546 anti-goat (catS: red) and Alexa Fluor 488 anti-mouse (nidogen-1: green). Magnification (insert) of the lower panel of the image (x 3). Arrowheads indicate cells containing catS and nid-1 close together (yellow). Scale bar = 25 µm. (F) BM matrix (5 µg) was incubated alone or with concentrated keratinocyte supernatant (10 µg protein) at pH 5.5 or 7.4 for 24 h. For controls, supernatants were pre-incubated for 30 min with E-64 or LHVS. Data represent immunoblot analysis with nid-1 antibodies from at least 3 independent experiments. (G) Pretreated lysates (10 µg) of wild-type C57/Bl6 mouse spleen (wt, lysates: 1–3) and catS-deficient spleen (<i>ctss^−/−</i>, lysates: 4–6) were incubated (pH 7.4, 37°C) for 1 h in order to inactivate all cysteine cathepsins, except catS. BM matrix (10 µg) was then incubated alone or with cell lysates (pH 7.4, 37°C) for 18 h. Immunoreactive nid-1 was revealed by western blot. Assays were performed on different samples (n = 15), which yielded similar results and a representative of two independent experiments is presented.</p

Hydrolysis of nid-2 by catS.

<p>(A) Western-blot of human nid-2 incubated with catS, catB, catL and catK at pH 5.5 for 30 min (enzyme:substrate ratio = 1∶143, w/w). (B) Densitometry analysis of nid-2 after incubation with catS (1, 5, 10, 30 min) at pH 5.5 (white bar) and pH 7.4 (grey bar). E-64 inactivated catS was used as a control. The percentage of residual nid-2 was expressed as means ± S.D (*, <i>P</i><0.05 and **, <i>P</i><0.01 when compared to control). A representative of three independent western blots at both pH is shown.</p

Cleavage of Nidogen-1 by Cathepsin S Impairs Its Binding to Basement Membrane Partners

<div><p>Cathepsin S (catS), which is expressed in normal human keratinocytes and localized close to the dermal-epidermal junction (DEJ) degrades some of major basement membrane (BM) constituents. Among them, catS readily hydrolyzed in a time and dose dependent manner human nidogen-1 (nid-1) and nidogen-2, which are key proteins in the BM structure. CatS preferentially cleaved nid-1 at both acid and neutral pH. Hydrolysis of nid-1 was hampered in murine <em>ctss</em>^−/− spleen lysates pretreated with inhibitors of other classes of proteases. Nid-1 was cleaved within its G2 and G3 globular domains that are both involved in interactions with other BM components. Binding assays with soluble and immobilized ligands indicated that catS altered the formation of complexes between nid-1 and other BM components. Assuming that the cleavage of nid-1 impairs its ability to crosslink with BM partners and perturbs the viscoelastic properties of BM matrix, these data indicate that catS may participate in BM proteolysis, in addition to already identified proteases.</p> </div

Influence of catS on the visco-elastic properties of BM matrix.

<p>(A) G’ (elasticity: black symbol) and G” (viscosity: white symbol) moduli of BM matrix (ECM gel: 8 mg/ml) without (C: control, triangles), or with catS (60 nM, squares) were determined by rheometry. A representative of three independent experiments is shown. (B) Western blot analysis of major BM constituents of ECM gel (nid-1, perlecan, laminin and collagen IV) with or without catS.</p