Our long-term goal is understanding the role of basement membranes (BM) in tissue organization and remodeling. With this application, we propose to continue our studies on laminin-5 (Ln-5), a BM extracellular matrix (ECM) component, its receptors, integrins a3bl and a6b4, and matrix metalloproteases (MMP) that induce migration by cleaving Ln-5. Concerted action of these molecules is critical for adhesion and migration of BM-contacting cells, and for proper organization of epithelial tissues, including epidermis, gut and breast. Our results will be relevant to several aspects of human health, such as wound healing, tissue regeneration and cancer invasion. In the past granting period, we made two key findings. The first is that a Ln-5 domain, a3LG3, binds integrin a3bl. The second is that membrane (MT1 -MMP) and secreted (MMP2) MMP can induce migration by cleaving Ln-5 at two sites on the g2 subunit. These findings begin to solve important longstanding issues in cell adhesion and migration. To take them to the next level of understanding, our research plan is articulated in three synergistic Specific Aims.
In Aim 1 we will perform in-depth characterization of the ligand-receptor interaction between Ln-5 a3LG3 domain and integrin a3bl, by using adhesion and migration assays and biochemical analyses with recombinant LG3 domains altered by site-directed mutagenesis. An important goal is to minimize the LG3 structure interacting with a3bl, and produce small recombinant or synthetic polypeptides to dissect adhesion and migration cellular responses to Ln-5. These same approaches will be extended to other possible integrin/LG domain interactions.
In Aim 2 we will investigate signaling initiated by Ln-5 or its integrin-binding modules, emphasizing cytoskeleton structural or regulatory molecules involved in adhesion and motility related structures such as focal contacts, filopodia, lamellipodia and ruffle borders. Both microscopy and biochemical approaches will be used. The goal is to dissect and correlate Ln-5 initiated signaling pathways to Ln-5 integrin-binding structures.
In Aim3 we will investigate molecular mechanisms for stimulation of migration by MMP cleavage of Lu-5. The rationale is that this process alters integrins/Ln-5 interactions, so that cells become migratory. To define the fine molecular details of this process, we will test four hypotheses, build outcome-based models and verify their predictions with functional assays and recombinant DNA approaches. Hopefully, with these Aims we will be able to outline a coherent picture as to how Ln-5, integrins and MMP interact to regulate cell adhesion and migration in the context of tissue organization and remodeling.
|Hintermann, Edith; Yang, Neng; O'Sullivan, Deirdre et al. (2005) Integrin alpha6beta4-erbB2 complex inhibits haptotaxis by up-regulating E-cadherin cell-cell junctions in keratinocytes. J Biol Chem 280:8004-15|
|Koshikawa, Naohiko; Minegishi, Tomoko; Sharabi, Andrew et al. (2005) Membrane-type matrix metalloproteinase-1 (MT1-MMP) is a processing enzyme for human laminin gamma 2 chain. J Biol Chem 280:88-93|
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|Schenk, Susann; Hintermann, Edith; Bilban, Martin et al. (2003) Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution. J Cell Biol 161:197-209|
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|Hintermann, Edith; Haake, Susan Kinder; Christen, Urs et al. (2002) Discrete proteolysis of focal contact and adherens junction components in Porphyromonas gingivalis-infected oral keratinocytes: a strategy for cell adhesion and migration disabling. Infect Immun 70:5846-56|
|Seftor, R E; Seftor, E A; Koshikawa, N et al. (2001) Cooperative interactions of laminin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma. Cancer Res 61:6322-7|
|Hintermann, E; Bilban, M; Sharabi, A et al. (2001) Inhibitory role of alpha 6 beta 4-associated erbB-2 and phosphoinositide 3-kinase in keratinocyte haptotactic migration dependent on alpha 3 beta 1 integrin. J Cell Biol 153:465-78|
|Kiosses, W B; Hahn, K M; Giannelli, G et al. (2001) Characterization of morphological and cytoskeletal changes in MCF10A breast epithelial cells plated on laminin-5: comparison with breast cancer cell line MCF7. Cell Commun Adhes 8:29-44|
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