The extracellular matrix (ECM) has been studied primarily for its ability to promote cell adhesion. It is becoming apparent that ECM components can regulate cell adhesion in a negative manner as well. Negative modulation of cell adhesion is important in events such as proliferation, development, wound healing, and metastasis in which changes in cell shape and increased motility are critical. Studies by myself and others indicate that thrombospondin (TSP) and tenascin (TN) are two such ECM molecules which destabilize cell adhesion. This proposal seeks a better understanding of how TSP and TN regulate cell adhesion, especially in bovine aortic endothelia cells.
The first aim of this proposal is to determine the mechanism whereby TSP modulates focal adhesions. Initial studies will determine if TSP is present at adhesion plaques to see if adhesions are accessible to TSP. TSP heparin-binding and non-binding fragments, alone and in combination, and synthetic peptides will be tested in focal adhesion assays for activity to determine which domains of TSP are active. Since TSP is clustered at the cell surface and endocytosed, a cross-linked fusion protein of the heparin-binding domain linked to Protein A will be used to determine if multivalency of this domain is required for TSP activity. TSP's effect on metabolism of cell surface proteoglycans will be assessed by cetyl pyridinium chloride precipitation studies to determine if TSP-cell binding causes loss of cell surface HSPGs, potentially disrupting focal adhesions. Time-lapse IRM will be used to determine if TSP prevents the re-formation of focal adhesions or causes their disassembly. Focal adhesion assays on synchronized cells will determine if the effect of TSP is cell cycle specific.
The second aim of this proposal is to probe the functional significance of TSP modulation of focal adhesions by studying the effect of TSP on cell migration, proliferation and DNA synthesis, and on strength of attachment.
The third aim i s to characterize the TN-mediated loss of adhesions in comparison to TSP. TN will be studied for its cell and substrate specificity and the ability of a C-6-S proteoglycan to neutralize TN activity. Monoclonal antibodies to domains of TN will help assess which parts of TN are active. These studies have potential significance for the study of vascular disease, particularly in reendothelialization of vessels and in the development of atherosclerotic disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL044575-04
Application #
3363383
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1990-07-01
Project End
1995-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
4
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Alabama Birmingham
Department
Type
Schools of Dentistry
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
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Orr, A Wayne; Murphy-Ullrich, Joanne E (2004) Regulation of endothelial cell function BY FAK and PYK2. Front Biosci 9:1254-66
Kevil, Christopher G; Orr, A Wayne; Langston, Will et al. (2004) Intercellular adhesion molecule-1 (ICAM-1) regulates endothelial cell motility through a nitric oxide-dependent pathway. J Biol Chem 279:19230-8
Orr, Anthony Wayne; Pallero, Manuel Antonio; Xiong, Wen-Cheng et al. (2004) Thrombospondin induces RhoA inactivation through FAK-dependent signaling to stimulate focal adhesion disassembly. J Biol Chem 279:48983-92
Elzie, Carrie Ann; Murphy-Ullrich, Joanne E (2004) The N-terminus of thrombospondin: the domain stands apart. Int J Biochem Cell Biol 36:1090-101
Orr, A Wayne; Elzie, Carrie A; Kucik, Dennis F et al. (2003) Thrombospondin signaling through the calreticulin/LDL receptor-related protein co-complex stimulates random and directed cell migration. J Cell Sci 116:2917-27
Orr, Anthony Wayne; Pedraza, Claudio E; Pallero, Manuel Antonio et al. (2003) Low density lipoprotein receptor-related protein is a calreticulin coreceptor that signals focal adhesion disassembly. J Cell Biol 161:1179-89
Orr, Anthony Wayne; Pallero, Manuel Antonio; Murphy-Ullrich, Joanne E (2002) Thrombospondin stimulates focal adhesion disassembly through Gi- and phosphoinositide 3-kinase-dependent ERK activation. J Biol Chem 277:20453-60
Goicoechea, Silvia; Pallero, Manuel Antonio; Eggleton, Paul et al. (2002) The anti-adhesive activity of thrombospondin is mediated by the N-terminal domain of cell surface calreticulin. J Biol Chem 277:37219-28
Murphy-Ullrich, J E (2001) The de-adhesive activity of matricellular proteins: is intermediate cell adhesion an adaptive state? J Clin Invest 107:785-90

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