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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