Defective laminin 5 processing in cylindroma cells

Abstract

Cylindromas are benign skin tumors occurring as multiple nodules characteristically well circumscribed by an excess of basement membrane-like material. To determine the molecular defects leading to extracellular matrix accumulation, the ultrastructural, immunological, and biochemical properties of cylindroma tissue and isolated cells were analyzed. In cylindromas, hemidesmosomes are reduced in number, heterogeneous and immature compared to the normal dermal-epidermal junction. Expression of the alpha6beta4 integrin in tumor cells is weaker than in basal keratinocytes of the epidermis. Moreover, although in the epidermis alpha2beta1-integrin expression is restricted to the basal cell layer, it is found in all neoplastic cells within the nodules. Laminin 5 is present throughout the whole thickness of the basement membrane-like zone whereas laminin 10 is restricted to the interface adjacent to the tumor cells. Furthermore, laminin 5 is not properly processed and most of the alpha3A and gamma2 laminin chains remain as 165-kd and 155-kd polypeptides, respectively. Mature laminin 5 is thought to be necessary for correct hemidesmosome and basement membrane formation and its abnormal processing, as well as the low expression of alpha6beta4 integrins, could explain the lack of mature hemidesmosomes. Together, the results show that multiple molecular defects, including alteration of laminin 5 and its integrin receptors, contribute to structural aberrations of the basement membrane and associated structures in cylindromas.

Figure 1.

Structural organization of laminin 5 and epitope mapping of the antibodies used in the study. During the processing of the laminin α3A chain, domains LG4 and LG5 are first removed (165-kd form), followed by removal of most of domain IIIa (145-kd form). Conversion of the 155-kd γ2 chain into a 105-kd polypeptide involves removal of domains LE1-3 and L4m. The domains removed during laminin 5 processing are indicated by stripped lines.

Figure 2.

Ultrastructure of the basement membrane-like zone surrounding cylindroma nodules in comparison to the epidermal basement membrane. A: Basement membrane-like zone bordering tumor cells. The thickness of the basement membrane (BM) is indicated by a line with arrow at both ends. Note the presence of electron-dense fibrils and filaments (white arrowheads) within the basement membrane-like zone. B: The basement membrane (BM) at the dermal-epidermal junction is indicated by an arrowhead. Small arrows point to the invaginations formed by hemidesmosomes at the ventral surface of basal keratinocytes of the normal epidermis. Note that such invaginations are not seen in A.

Figure 3.

Ultrastructural alterations and heterogeneity of hemidesmosomes in cylindroma cells (B–E) compared to normal hemidesmosomes of basal keratinocytes of the epithelium (A). Hemidesmosomes (HD), lamina densa (LD), and anchoring fibrils (AF) are marked. Note in B to E the heterogeneous appearance of tumor cell hemidesmosomes and absence of a distinct lamina densa.

Figure 4.

Indirect immunofluorescence staining of laminins and nidogen in cylindromas (A–C) and at the dermal-epidermal junction (D–F). Cryosections (5 μm) of tissues were incubated with mouse monoclonal antibodies BM165 (A and D) or 4C7 (B and E) against the human laminin α3 and α5 chains, respectively, or with a polyclonal serum against nidogen 1 (C and F). Mouse or rabbit immunoglobulins were detected with Cy3-conjugated secondary antibodies. For each different staining, the pictures corresponding to the cylindroma islands (A–C) or to the dermal-epidermal junction are from the same tissue section. Immunoreactivity corresponding to the laminin α3 chain is observed in the whole thickness of the material surrounding the tumor islets (A). By contrast, immunoreactivity corresponding to the laminin α5 chain (B) or to nidogen (C) is mainly restricted to the inner face of the basement membrane outlining the cylindromas, and for α5 to some intranodular deposits.

Figure 5.

Unprocessed laminin 5 is present in cylindroma tissue. Cryosections of biopsied tissue were stained by indirect immunofluorescence with antibodies specific for different domains of the laminin α3A and γ2 chains as indicated on the figure. Staining with γ2 chain-specific antibodies indicate that domain L4m is retained in the laminin 5 deposited around cylindroma (A) but that it has been removed at the dermal-epidermal junction (E) whereas domains LE4-6 are present at both locations (B and F). Anti-α3AIIIa stains the basement membrane area around cylindromas (D) and at the dermal-epidermal junction (H). Anti-α3LG4-5 does not decorate specific structures at the dermal-epidermal junction (G), which indicates that the carboxy-terminal end of the α3A chain has been processed at this location. By contrast, it decorates with a thin line the periphery of the tumor nests and stains intranodular deposits reflecting the presence of unprocessed α3A chain within the tumor islands (C).

Figure 6.

Altered processing of laminin 5 in primary cultures of cylindroma cells. The culture medium of cylindroma cells (Cy) and of primary human keratinocytes (Ke) was mixed 1:1 with Laemmli buffer and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions on 4 to 10% acrylamide gels (50 μl/lane). After transfer to nitrocellulose membranes, the blots were incubated with mouse monoclonal antibodies BM165 (A) or D4B5 (B) against the human laminin α3 or γ2 chains, respectively. The blot shown in B was extensively washed and reblotted with a goat antibody against a carboxy-terminal peptide of the laminin β3 chain (C). Bound antibodies were detected using horseradish peroxidase-conjugated secondary antibodies followed by enhanced chemiluminescence. Molecular weight markers are indicated at the right of the blots. Arrowheads at the left of the blots indicate the migration mobility of the different laminin chains. The relative optical density of the bands corresponding to differently processed forms of the laminin α3A and γ2 chains was measured by scanning densitometry. Each column represents the relative percentage of the processed (dots) and unprocessed (stripes) forms of the laminin α3A (D) and γ2 (E) chains shown in blots A and B, respectively.

Figure 7.

Integrin expression in cylindroma cells and basal keratinocytes of the epidermis. Tissue cryosections were processed for indirect immunofluorescence staining with antibodies against the integrin β1 (A–C), β4 (D–F), α2 (G–I), α3 (J–L), and α6 (M–O) chains. Left: Overviews of the staining of cylindroma nodules (low magnification) (A, D, G, J, M). With each antibody, higher magnification of the stainings observed for tumor cells (B, E, H, K, N) and basal keratinocytes (C, F, I, L, O) were recorded on the same tissue section.