1985 — 2001 |
Couchman, John R. |
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. |
Basement Membrane Proteoglycans @ University of Alabama At Birmingham
Basement membranes are specialized zones of connective tissue underlying parenchymal cells and separating them from supporting connective tissues. They are composed of a number of macromolecules which interact with each other and with cell surfaces. Functionally they not only provide support and a selective filter but, through cell surface interactions, profoundly influence cell behavior such as growth, migration and differentiation. Heparan sulfate proteoglycan has been identified as an important basement membrane constituent and is implicated in control of basement membrane permeability. We have also identified and partially characterized a basement membrane-specific chondroitin sulfate proteoglycan synthesized by the PYS-2 cell line. Antibodies have been raised against this and the heparan sulfate proteoglycan, purified from bulk cultures, and these stain many mammalian basement membranes. In this proposal we aim to further characterize the proteoglycans and survey their distribution in skin and other basement membranes, with particular emphasis on the embryonic development and ultrastructural studies. The antisera already raised will be exhaustively characterized and monoclonal antibodies will be raised against the PYS-2 chondroitin sulfate proteoglycan. These will assist in immunohistochemical and structural analysis of this newly recognized basement membrane component. The intermolecular interactions between the PYS-2 proteoglycans and other basement membrane macromolecules will also be investigated. It is intended that data concerning the role of basement membrane proteoglycans in the dermal-epidermal junction will be gained which may be of future significance in the study of a wide range of skin diseases where lesions at the basement membrane are an integral part of the pathological process.
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1989 — 1993 |
Couchman, John R. |
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. |
Fibronectin and Skin Development @ University of Alabama At Birmingham
This proposal concerns the molecular basis by which fibronectins influence maturation in the skin. This family of closely related glycoproteins is abundant in mammalian skin throughout development and postnatal life, especially within the dermis, the connective tissue sheaths of nerve, blood vessel and muscle fibers within the dermis and the dermal-epidermal junction and other basement membranes. Previous data suggest that fibronectins, through their ability to influence cellular behavior, are important extracellur matrix molecules. They can specifically promote cell-matrix adhesion and facilitate migration, processes inherently involved in skin development and repair. The molecular control of these processes is, however, not well understood. Since fibronectins are multi- domain molecules, able to interact with collagens, proteoglycans and cell surfaces we hypothesize that it is the controlled interaction of its constituent domains which modulate adhesive responses to fibronectins, to allow migration or firm anchorage. Through the use of purified fibronectin domains, specific antibodies, synthetic peptide analogues of fibronectin and analysis of cell surface molecules potentially involved in modulating cellular interactions with fibronectin, we shall examine how adhesion and migration are controlled in cultured dermal fibroblasts. Fibronectin is synthesized very early in development and is enriched in embryonic basement membranes. However, its distribution with in the human dermal-epidermal junction and changes which may occur in concert with epidermal maturation are unknown. Since many severe skin diseases involve lesions in the basement membrane, documentation of its composition through development is important. We shall use immunoelectron microscopy to map fibronectins in the dermal-epidermal junction. Moreover, our data with an epithelial cell line show that fibronectins may promote basement membrane assembly, a process commensurate with tissue organization and repair. Presently, the molecular mechanism of its action is unknown and we propose to test whether fibronectins 'nucleate' assembly of the basement membrane matrix and/or influence epithelial behavior and promote direct export of matrix components.
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1994 — 1997 |
Couchman, John R. |
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
Cellular interactions with extracellular matrix molecules are known to be involved in may developmental, repair, and homeostatic processes, but only recently has it been established that these interactions initiate transmembrane signalling events. The most well studied pathways to date involve tyrosine phosphorylations initiated by Beta1 and Beta3 integrin receptor-ligand interactions. the adhesion of most anchorage-dependent cells in culture, and some in vivo, is characterized by structures known as focal contacts or focal adhesions. These structures are thought to be integral to wound healing processes was well as providing stable anchorage for normal cells, but are often change in structure and composition in transformed cells, where they may even be absent. They are also an excellent model for transmembrane signalling. While many focal adhesion constituents are known, the process of assembly is not understood. Both tyrosine phosphorylation and subsequent protein kinase C (PKC) activity may be involved in their formation and in this respect, the pathways resemble those for some growth factor and lymphocyte receptor interactions. The long term goal of this research is to understand the molecular events leading to the formation and maintenance of focal adhesions in primary cultured cells. It is proposed that cells forming adhesions on a fibronectin substrate utilize a dual receptor system involving both integrins and cell surface proteoglycans. The latter may promote PKC activity and the assembly of focal adhesions. The transmembrane proteoglycan involved in this process has recently been identified as a member of the syndecan family of proteoglycans.
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1998 — 2000 |
Couchman, John R. |
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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