Laminin-5 (LN5) is a matrix component of epithelial tissue basement membranes and plays an important role in the initiation and maintenance of epithelial cell anchorage to the underlying connective tissue. Here we show that two distinct LN5 function-inhibitory antibodies, both of which bind the globular domain of the ␣3 subunit, inhibit proliferation of epithelial cells. These same antibodies also induce a decrease in mitogenactivated protein kinase activity. Inhibition of proliferation by the function-perturbing LN5 antibodies is reversed upon removal of the antibodies and can be overcome by providing the antibody-treated cells with exogenous LN5 and rat tail collagen. Because epithelial cells use the integrin receptor ␣3␤1 to interact with both LN5 and rat tail collagen, we next investigated the possibility that integrin ␣3␤1 is involved in mediating the proliferative impact of LN5. Proliferation of human epithelial cells is significantly inhibited by a function-perturbing ␣3 integrin antibody. In addition, antibody activation of ␤1 integrin restores the proliferation of epithelial cells treated with LN5 functionperturbing antibodies. These data indicate that a complex comprising LN5 and ␣3␤1 integrin is multifunctional and contributes not only to epithelial cell adhesion but also to the regulation of cell growth via a signaling pathway involving mitogen-activated protein kinase. We discuss our study in light of recent evidence that LN5 expression is up-regulated at the leading tips of tumors, where it may play a role in tumor cell proliferation. INTRODUCTIONCell interaction with elements of the extracellular matrix impacts their adherence, motility, as well as protein and gene expression (for example, see Adams and Watt, 1993;Roskelly et al., 1995). In intact normal tissue epithelial cells bind to extracellular matrix molecules, which are organized into a complex multiprotein structure called the basement membrane. The major components of the latter include type IV collagen, proteoglycans, and laminins. One laminin isoform, laminin-5 (LN5), 1 in particular plays an important role in establishing firm adherence of epithelial cells to the basement membrane, because it is necessary for the assembly and maintenance of stable anchorage devices between epithelial cells and matrix called hemidesmosomes (Baker et al., 1996;Green and Jones, 1996). However, recent reports also indicate that LN5 is expressed at the budding tips of invading tumor cells, i.e., at sites where cancer cells are undergoing cell division but where there are most likely no hemidesmosomes (Pyke et al., 1994(Pyke et al., , 1995. This provides an indication § Corresponding author. E-mail address: j-jones3@nwu.edu. 1 Abbreviations used: BrdU, bromodeoxyuridine; DAPI, 4,6-diamidino-2-phenylindole; EGF, epidermal growth factor; FN, fibronectin; hLN5, human laminin-5; Ig, immunoglobulin; LN, laminin; MAP, mitogen-activated protein; MAPK, MAP kinase; RTC, rat tail collagen; rtLN5, rat laminin-5.© 1999 by The American Society for Cell Biology 259 that L...

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Interactions between extracellular matrix (ECM) proteins and prostate carcinoma cells provide a dynamic model of prostate tumor progression. Previous work in our laboratory showed that laminin‐5, an important member of a family of ECM glycoproteins expressed in the basal lamina, is lost in prostate carcinoma. Moreover, we showed that the receptor for laminin‐5, the α6β4 integrin, is altered in prostate tumors. However, the genes that laminin‐5 potentially regulates and the significance of its loss of expression in prostate cancer are not known. We selected cDNA microarray as a comprehensive and systematic method for surveying and examining gene expression induced by laminin‐5. To establish a definitive role for laminin‐5 in prostate tumor progression and understand the significance of its loss of expression, we used a cDNA microarray containing 5289 human genes to detect perturbations of gene expression when DU145 prostate carcinoma cells interacted with purified laminin‐5 after 0.5, 6, and 24 h. Triplicate experiments showed modulations of four, 61, and 14 genes at 0.5, 6, and 24 h, respectively. Genes associated with signal transduction, cell adhesion, the cell cycle, and cell structure were identified and validated by northern blot analysis. Protein expression was further assessed by immunohistochemistry. Mol. Carcinog. 30:119–129, 2001. © 2001 Wiley‐Liss, Inc.

Hemidesmosomes are complex macromolecular structures which integrate elements of the extracellular matrix and the cytoskeleton of epithelial cells. To characterize cell-matrix interactions in the hemidesmosome, we have made use of 804G cells which possess the unusual ability to assemble hemidesmosomes in vitro. During the course of our studies, we have raised a set of monoclonal antibodies against rat laminin-5, the major structural element comprising 804G matrix. One of these, termed CM6, recognizes the 150 kDa alpha chain of rat laminin-5 and binds the globular (G) domain of intact laminin-5 molecules as determined by rotary shadowing. CM6 antibodies perturb formed hemidesmosomes in 804G cells. In particular, within 1 hour of incubation of 804G cells with CM6 antibodies, colocalization of laminin-5 and alpha 6 beta 4 integrin is lost and by 2 hours, staining generated by hemidesmosomal antibodies appears primarily cytoplasmic in the perinuclear zone. Ultrastructurally, CM6 antibodies first appear to induce detachment of hemidesmosomes from the underlying matrix. Next, portions of the basal cell surface invaginate to form vesicles whose cytoplasmic-facing surface is coated with hemidesmosomes still associated with keratin intermediate filaments. Anchoring filaments extend into the inside compartment of the vesicles. We have also studied the impact of CM6 antibodies on a model system in which the matrix of 804G cells induces de novo assembly of hemidesmosomes in human keratinocytes. This process involves the plasma membrane reorganization of the hemidesmosome associated integrin alpha 6 beta 4 as well as a redistribution of other hemidesmosome components such as the 230 kDa bullous pemphigoid antigen. Pretreatment of 804G matrix with CM6 antibodies blocks such plasma membrane reorganization of hemidesmosome components and inhibits hemidesmosome formation. Our studies indicate a crucial role for the G domain of the alpha chain of laminin-5 in both nucleation of hemidesmosome assembly as well as maintenance of hemidesmosome structural integrity.

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Laminin-5r is a basement membrane component that promotes rapid adhesion and hemidesmosome formation in epithelial cells. We raised monoclonal antibodies and identified their corresponding epitopes on the constituent chains of laminin-5r by western blotting. Using a combination of immunoprecipitation and ELISA assays, we determined that these epitopes are differentially exposed on two forms of the laminin-5r heterotrimer: soluble (passively adsorbed onto plastic) and cell-associated. Antibody 5C5 epitope is exposed on the cell-associated form, but not the soluble/passively adsorbed form of laminin-5r. Epitopes reactive with antibodies CM6, FM3, and TR1 are also preferentially exposed on cell-associated laminin-5r, such that reactivity of these antibodies with the cell-associated form is fourfold higher than with the soluble/passively adsorbed form in ELISA assays. Incubation of passively adsorbed laminin-5r with the human epithelial cell line SCC12 induced exposure of 5C5 and CM6, FM3, or TR1 epitopes. These data suggest that cells actively modify laminin-5r, perhaps during matrix assembly, and that the 5C5 epitope may serve as a marker for assembled laminin-5r matrix.

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