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Cell Migration in Tissue Remodeling and Cancer

V. Quaranta, F. Frasier, S.Z. Domanico, G. Giannelli, N. Koshikawa, L. Mullen, T.A. Romano, S. Schenk, M. Shang, D. Richards*

* University of California, San Francisco, CA

Cellular spatial organization within tissues is fundamental to both health and disease. The extracellular matrix (ECM), the matter positioned in between cells, contains important spatial cues for cells. Although these cues are poorly understood, it is known that they can both stimulate and constrain migration of cells across tissues. The structural nature of ECM cues is the subject of intensive investigation, because these cues may hold the key to intervention in many disease processes, including tumor invasion and metastasis.

We focus on a specialized type of ECM, the basement membrane, and its interaction with a particular cell type, the epithelial cell. Epithelial cells and basement membranes make up a large part of organs and tissues in the body (e.g., mammary gland, skin, gum, liver, kidney, intestine) and give rise to most human cancers (carcinomas).

REGULATION OF CELL MIGRATION BY LAMININ-5

The basement membrane of many epithelial tissues, such as mammary glands, gut, and epidermis, contain the ECM molecule laminin-5. Genetic evidence indicates that laminin-5 is critical to the function of basement membranes in these tissues. Inherited mutations in the genes for the subunits of laminin-5 can cause severe disruption of epithelial organization, such as extensive skin blistering, often with fatal consequences. Receptors for laminin-5 are members of the integrin family. Mutations in these receptors cause similar phenotypic changes. Thus, laminin-5 in conjunction with its receptors is critical to epithelial organization and function.

We showed that laminin-5 can alter the balance between static adhesion and migration in breast epithelial cells in contact. The matrix metalloprotease MMP2 changes laminin-5 from a static to a migratory substrate by cleaving the short arm of the 2 subunit of the laminin. We localized the cleavage site by amino-terminal sequencing and are investigating the molecular basis of this promigratory mechanism. Preliminary results suggest that the MMP2 cleavage causes conformational changes in the structure of the putative cell-attachment site on the ₃ subunit of laminin-5. If confirmed, this observation could produce exciting insights into the mechanisms of cell adhesion to laminins. Adhesion and migration on laminins is not as well understood as it is on other ECM components, such as fibronectin and collagen. The mechanism of action of laminins may differ from that of other ECM components.

MMP2-cleaved laminin-5 is easily detected by the presence of a 2 chain with reduced molecular weight, which we named 2x. Screening of mammary gland tissue at distinct stages of the reproductive cycle showed that the presence of 2x correlated well with the occurrence of tissue remodeling in the gland. For example, cleaved laminin-5 is present during pregnancy and after lactation but is absent before puberty and during lactation. These data suggest that MMP2 cleavage of laminin-5 may be intimately involved with the blooming of new buds in the ducts.

Another mechanism of migration on laminin-5 involves integrin activation. A receptor for laminin-5, the integrin ₃ß₁, promotes both static adhesion and migration of breast epithelial cells on the laminin. We found that the shift from static adhesion to migration requires activation of this receptor. In attempting to define what ₃ß₁ activation means in molecular terms, we detected a signaling pathway that depends on the interaction of this integrin with heterotrimeric G proteins. The invasive properties of breast cancer cells can be explained, in part, by our data.

The integrin ₆ß₄ supports static adhesion of epithelial cells to laminin-5. Cells dramatically upregulate the affinity and avidity of the interaction of ₆ß₄ with laminin-5, and we propose that this mechanism is critical to the formation of organized epithelial sheets. The molecular details of this interaction, which may also be relevant to tissue remodeling and tumor invasion, are under investigation.

In summary, several distinct mechanisms may cooperate to regulate the balance between static adhesion and migration on laminin-5. The challenge is to understand these mechanisms and to develop technologies that can be used to manipulate tissue remodeling and tumor invasion.

THE TOOTH-GINGIVAL INTERFACE

We are using the soft gum tissue surrounding the tooth as a model to explore the role of laminin-5 and its receptors in epithelia. Junctional epithelial cells of periodontal tissue adhere to the tooth surface via a specialized basement membrane, the IBL. Our data show that laminin-5 is the main component of IBL. In vitro, epithelial cells adhere preferentially to those parts of the tooth surface that are coated by laminin-5. Most likely, the interaction between junctional cells and laminin-5 provides the architectural cues to form the seal between the tooth and gum that isolates the inner bone and connective tissues from the oral cavity. Formation and repair of this seal probably require regulation of tight adhesion and migration.

Factors that participate in these regulatory events may be similar or identical to those that act in more complex epithelial organs. Unique among basement membranes, the IBL coats a hard surface (the tooth enamel) and is not in contact with soft connective tissues that add complexity to the system. This characteristic may make it easier to characterize regulatory events and to determine the molecular determinants in the IBL, particularly those specified by laminin-5, that the gum epithelium recognizes as organizational cues. We are testing these possibilities in tooth organotypic cultures in vitro in which we can manipulate the formation and disruption of the periodontal seal between oral keratinocytes and the tooth surface.

INFLUENCE OF TETRASPANINS ON INTEGRIN-DEPENDENT MIGRATION

The mechanical aspects of migration are carried out by receptors of the integrin type, which propel cell movement and directly engage with the ECM. We detected a mechanism whereby the integrin ₆ß₁, a receptor for laminins, can regulate cell migration without engaging with ECM migratory substrate. Our results suggest that this mechanism requires the interaction of ₆ß₁ with CD81, a member of the growing family of tetraspanins.

The proposed function for tetraspanins, serpentine cell-surface molecules with 4 membrane-spanning domains, is that of docking molecules that promote the formation of multireceptor clusters on the cell surface. Thus, integrins may associate with other cell-surface receptors via tetraspanins. A physical association of integrins with several distinct tetraspanins has been proved. We are particularly interested in tetraspanins that may interact with the laminin receptors ₆ß₁ and ₃ß₁. Preliminary findings suggest that the outside-in signaling initiated by integrins may differ distinctly, depending on differences in tetraspanin interactions.

PUBLICATIONS

Faccio, R., Grano, M., Colucci, S., Zambonin-Zallone, A., Quaranta, V., Pelletier, A.J. Activation of _vß₃ integrin on human osteoblast-like cells stimulates adhesion, migration and intracellular calcium mobilization. Biochem. Biophys. Res. Commun. 249:522, 1998.

Falk-Marzillier, J., Pelletier, A.J., Mullen, L., Domanico, S.Z., Quaranta, V. Characterization of molecular complex between integrin ₆ß₄ and laminin-5 deposited by 804G epithelial cells. Biochem. Biophys. Res. Commun., in press.

Fitchmun, M.I., Marshall, E., Cruz, G., Falk-Marzillier, J., Jones, J.R.J., Quaranta, V. Mode of adsorption and orientation of an extracellular matrix protein affect its cell adhesion promoting activity. Anal. Biochem., in press.

Kiosses, B., Klaus, M.H., Giannelli, G., Quaranta, V. Characterization of laminin-5 induced morphological and cytoskeletal changes in MCF10A breast epithelial cells. Cell Motil. Cytoskeleton, in press.

Mullen, L., Richards, D.W., Quaranta, V. Evidence that laminin-5 is a component of the tooth surface internal basal lamina supporting epithelial cell adhesion. J. Periodontal Res., in press.

Plopper, G.E., Kiosses, W.B., Cirulli, V., Domanico, S.Z., Quaranta, V. Migration of breast epithelial cells on laminin-5: Differential role of integrins in normal and transformed cell types. Breast Cancer Res. Treat., in press.

Romano, T.R., Ridgway, S.H., Felten, D.L., Quaranta, V. Molecular cloning and characterization of CD4 in an aquatic mammal, the white whale, Delphinapterus leucas. Immunogenetics, in press.