1997 — 1999 |
Woods, Anne C. |
P60Activity Code Description: To support a multipurpose unit designed to bring together into a common focus divergent but related facilities within a given community. It may be based in a university or may involve other locally available resources, such as hospitals, computer facilities, regional centers, and primate colonies. It may include specialized centers, program projects and projects as integral components. Regardless of the facilities available to a program, it usually includes the following objectives: to foster biomedical research and development at both the fundamental and clinical levels; to initiate and expand community education, screening, and counseling programs; and to educate medical and allied health professionals concerning the problems of diagnosis and treatment of a specific disease. |
Feasibility Study--Syndecan 2 Proteoglycan and Matix Metabolism @ University of Alabama At Birmingham
The long term objective of this research is to understand the molecular basis controlling extracellular matrix assembly, leading to possibilities for intervention in those connective tissue diseases characterized by excessive matrix deposition. Fibrosis commonly occurs following inflammation, when connective tissue cells remain "activated." Despite much research, the molecular basis for matrix assembly at the cell surface is still not well understood. Cell culture studies have shown a clear role for integrins in fibronectin fibrillogenesis, an early and important step in this process. In addition, there are poorly understood roles for heparin-binding domains of fibronectin, and an intact microfilament cytoskeleton. Novel data now indicates that a cell surface heparan sulfate proteoglycan, syndecan 2, may be pivotal in matrix assembly. Transfected cells expressing syndecan 2 that lacks the terminal 14 amino acids in the cytoplasmic domain could not establish a pericellular matrix, unlike those transfected with the full length core protein, or wild-type or similarly truncated syndecan 4. Synthetic peptides encompassing this region of syndecan 2 were phosphorylated in vitro by protein kinase C, whose activation is needed for matrix fibrillogenesis. Signaling through the cytoplasmic domain of syndecan 2, may, therefore, play a major role in matrix metabolism. To test this, the proposed studies will: 1) confirm that a similar disruption of matrix assembly occurs when human dermal fibroblasts are transfected with truncated syndecan 2, and that this correlates with the expression level of the transfect product. 2) determine, by immunofluorescence and immunoprecipitation pulse-chase experiments, whether this is due to failure to externalize and/or bind fibronectin at the cell surface, or due to rapid internalization and degradation. 3) determine, by phosphorylation assays in vitro, and in cells containing normal or mutated syndecan 2, whether phosphorylation of the cytoplasmic domain mediates the signaling mechanism controlling matrix assembly. 4) determine, by FACS analysis, immunofluorescence, and binding assays, if altered metabolism correlates with integrin expression, usage or affinity resulting from expression of truncated syndecan 2.
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0.958 |
1998 — 2006 |
Woods, Anne C. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Syndecan-2 Proteoglycan Regulation of Matrix Assembly @ University of Alabama At Birmingham
The long-term goal of this research is to understand the mechanisms that control extracellular matrix assembly. This knowledge will allow future intervention in disease states where matrix deposition is excessive, a major problem in diabetes, where glomerulosclerosis and interstitial fibrosis contribute to renal failure. The use of dominant negative constructs has shown that syndecan-2 transmembrane heparan sulfate proteoglycan controls the assembly of extracellular matrix at the cell surface, the first stage in organized matrix deposition. Using cell biological, immunological, and molecular biology techniques, we will: 1) determine how syndecan-2 cytoplasmic domain controls matrix assembly, which regions of the cytoplasmic domain are involved, and whether this is regulated by phosphorylation events and/or binding to other cytoplasmic proteins; 2) determine how signaling through syndecan-2 occurs, whether the heparan sulfate glycosaminoglycans are involved or necessary, whether syndecan-2 activates the integrin family of matrix receptors and whether its acts via cytoskeletal organization; and 3) determine whether syndecan-2 protein levels are increased in diabetic nephropathy in concert with, or preceding, matrix deposition increases, whether protein and mRNA levels are elevated in response to mediators thought to promote diabetic nephropathy in vivo, such as transforming growth factor beta and high glucose, and whether transcription of the gene is under the control of similar mediators. We will directly test the hypothesis that syndecan-2 regulates matrix accumulation in kidney interstitial cells.
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0.958 |
2001 — 2006 |
Woods, Anne C. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Proteoglycan Mediated Signaling in Cell Adhesion @ University of Alabama At Birmingham
DESCRIPTION: Anchorage-dependent cells respond to matrix molecules by the formation of focal adhesions. These are sites of complex transmembrane signaling which impact cell behavior and growth, both through short term effects on cytoskeletal organization and longer term effects on gene transcription. The long term goal of this research is to understand the molecular mechanisms underlying focal adhesion formation and subsequent signaling role in migration, anchorage, and matrix assembly and turnover. Cell-matrix interactions have important roles in development and wound repair, and are altered in the pathogenesis of rheumatic diseases, tumorigenesis and fibrosis. Syndecan-4 is a transmembrane heparan sulfate proteoglycan that regulates focal adhesion assembly. Transfection studies show that a region of its cytoplasmic domain can influence focal adhesions, cytoskeletal organization, and migration. This region of syndecan-4, but not equivalent regions from the other three syndecan members, can directly bind protein kinase C and regulate its activity. Oligomerization of the core protein is required. This, and protein kinase C regulatory ability, are both augmented by syndecan-4 interactions with phosphatidylinositol 4,5 biphosphate (PIP2), known to be a regulator of actin filament and focal adhesion assembly. The proposed studies will determine: (1) whether protein kinase C, syndecan-4 and PIP2 form a ternary signaling complex in vivo, (2) the sites of interaction between these three cell membrane associated components and how this is regulated, (3) the roles of the ectodomain and cytoplasmic domain of syndecan-4 core proteins, and of its glycanation, in focal adhesion assembly, and (4) the pathway by which protein kinase C regulates focal adhesion assembly, possibly through convergence with other signaling cascades, such as G proteins of the Ras superfamily, lipid metabolism and tyrosine kinases. These studies will use a combination of physical, chemical, immunological and molecular techniques, and will serve as a basis for intervention in the controlling role of focal adhesion and cytoskeletal organization.
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0.958 |