Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-03-15
2003-05-20
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S252300, C435S069100, C435S069700, C530S395000
Reexamination Certificate
active
06566074
ABSTRACT:
BACKGROUND OF THE INVENTION
Adhesion of cells to their surrounding extracellular matrix (ECM) in vivo regulates their morphology, proliferation, migration, survival and differentiation. Adams et al. (1993)
Development
117:1183-1198; Ashkenas et al. (1996)
Dev. Biol
. 180:433-444; Giancotti (1997)
Curr. Opin. Cell. Biol
. 9:691-700; Howe et al. (1998)
Curr Opin. Cell Biol
. 10:220-231. In vitro, the interactions of cells with ECM molecules such as fibronectin result in cell attachment, spreading and the assembly of focal adhesions and actin stress fibers. Burridge et al. (1996)
Ann. Rev. Cell Dev. Biol
. 12:463-518. Focal adhesions are macromolecular complexes made up of transmembrane adhesion receptors and intracellular proteins with structural and signaling functions. Burridge et al., supra; Clark et al. (1995)
Science
268:233-239.
Two independent adhesion receptor-mediated signals are required for the assembly of these macromolecular complexes when cells are plated on fibronectin. One signal is mediated through integrins and involves the RGD-containing cell-binding domain of fibronectin. Hynes (1992)
Cell
69:11-25; Clark et al. (1995)
Science
268:233-239. The second signal is mediated through cell surface heparan sulfate proteoglycans (HSPGs) and involves heparin-binding domains of fibronectin. Woods et al. (1986)
EMBO J
. 5:665-670.
The formation of complete focal adhesions and stress fibers in the context of integrins has been shown to require integrin clustering, integrin occupancy, tyrosine phosphorylation and cytoskeletal integrity. Hynes (1992)
Cell
69:11-25; Clark et al. (1995)
Science
268:233-239; Miyamoto et al. (1995)
J. Cell Biol
. 131:791-805; Miyamoto et al. (1995)
Science
267:883-885. Integrin signaling pathways involve the small GTP-binding protein Rho. Parsons (1996)
Curr. Opin. Cell Biol
. 8:146-152; small GTP-binding protein Rho. Parsons (1996)
Curr. Opin. Cell. Biol
. 8:146-152; Tapon et al. (1997)
Curr. Topics Cell Biol
. 9:86-92; Clark et al. (1998)
J. Cell Biol
. 142:573-586.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery that syndecan-4 acts cooperatively with integrins in generating signals for cell spreading and for the assembly of focal adhesions and actin stress fibers. In addition, it was discovered that these joint signals are regulated in a Rho-dependant manner.
Specifically, it was found that when a cell, which does not express fibronectin, is plated solely on the cell binding domain of fibronectin or antibodies directed against the&bgr; integrin chain, the cell attaches but fails to spread or assemble focal adhesions or actin stress fibers. When antibodies directed against the ectodomain of syndecan-4 are then added, the cells spread fully and assemble focal adhesions and actin stress fibers which are indistinguishable from cells plated on fibronectin. Moreover, it was found that the activation of the GTP-binding protein Rho, which is a key regulator in adhesion-mediated signaling events through integrins, is also dependent on syndecan-4 signaling events.
Accordingly, in one aspect, the invention features, a method of modulating the spatial or positional relationship of a cell to a substrate, or modulating the intracellular response of a cell to a substrate, in vitro or in vivo. The spatial or positional relationship of a cell to a substrate refers to the spatial or positional relationship of one or more points on the cell to substrate, and includes, e.g., cell attachment, cell spreading, or cell migration. Cell spreading includes the movement of an entire cell across a substrate as well as the movement of one part of the cell but not another part of the cell with regard to the substrate. The intracellular response of a cell to a substrate refers the response, e.g., the movement or formation or disolution of, a cellular component, e.g., a cell membrane protein, or an actin stress fiber, to a substrate and includes the formation or dissolution of a focal adhesion.
The method includes administering an agent which modulates the interaction, e.g., the binding, of the syndecan-4 ectodomain with a counterligand, thereby modulating the spatial or positional relationship of a cell to a substrate, or modulating the intracellular response of a cell to a substrate. The preferred counterligand is an ECM component, e.g., the heparin-binding domain of a component of the extracellular matrix (ECM) such as fibronectin, vitronectin, a laminin or a collagen.
In a preferred embodiment, the agent inhibits syndecan-4 ectodomain binding to a counterligand, e.g., the heparin-binding domain of an ECM molecule. The spatial or positional relationship of a cell to a substrate, or the intracellular response of a cell to a substrate, can be inhibited or reduced by interfering with the interaction of syndecan-4 a counterligand, e.g., a heparin-binding domain of an ECM molecule. Agents which interfere with the binding of syndecan-4 to a counterligand include agents which bind to an ECM molecule, such as fibronectin, vitronectin, a laminin or a collagen, and thereby inhibit the binding of the ectodomain of syndecan-4, with a heparin-binding domain of the ECM molecule. The invention is not limited by the particular mechanism of inhibition. The agent can e.g., act: by binding and occupying the site normally bound by the ectodomain of syndecan-4, by binding to and changing the shape of the ECM molecule (such that counterligand binding is inhibited); or by binding an ECM molecule and sterically hindering binding of the ectodomain of syndecan-4 to a heparin-binding domain of the molecule. Examples of such agents include: a syndecan-4 protein, e.g., a soluble syndecan4 protein, or a heparin binding domain (HBD)-binding portion thereof, e.g., from amino acid residues 90 to 120 of SEQ ID NO:2; a fusion of a syndecan-4 protein, e.g., a fusion of syndecan-4, or a HBD-binding portion thereof, to another polypeptide, e.g., a polypeptide which promotes solubility or which targets or binds to a substrate; a polypeptide other than syndecan-4 which binds to an ECM molecule, e.g., to a heparin binding domain of an ECM molecule, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay; antibodies to an ECM molecule, e.g., antibodies which bind the site normally bound by the ectodomain of syndecan-4, antibodies which bind to and change the shape of the ECM molecule, or antibodies which bind and sterically hinder binding of the ectodomain of syndecan-4 to a heparin-binding domain of an ECM molecule. ECM molecules include, for example, fibronectin, vitronectin, laminins and collagens.
In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
The inhibitory agent can act by binding syndecan-4. While not wishing to be bound by theory these agents may bind to syndecan-4 but fail to provide, or otherwise inhibit the transduction of a signal which normally arises when syndecan-4 binds to a heparin-binding domain of an ECM molecule. These agents can e.g., bind and occupy the site on the ectodomain of syndecan-4 normally bound by a heparin-binding domain of an ECM molecule; bind to and change the shape of syndecan-4; or bind and sterically hinder binding of a heparin-binding domain of an ECM molecule to the ectodomain of syndecan-4. Examples of such agents include an analog of fibronectin or of a syndecan-4 binding portion of fibronectin; an analog of vitronectin or of a syndecan-4 binding portion of vitronectin; an analog of a laminin or of a syndecan-4 binding portion of a laminin; an analog of a collagen or of a syndecan-4 binding portion of a collagen; an analog of a soluble heparin binding domain; a fusion of all, or a syndecan-4 protein binding portion, of an analog of an EMC molecule to another polypeptide, e.g., a polypeptide which promotes solubility or which targets or binds to a substrate; a polypeptide other than a heparin binding domain which binds to the ectodomain of syndecan-4, e.g., a polypeptide selected for binding in, e.
Fish & Richardson PC
Katcheves Konstantina
The General Hospital Corporation
Yucel Remy
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