The central hypothesis of this proposal is that syndecan-2, a transmembrane heparan sulfate proteoglycan is a mediator of renal tubulo-interstitial fibrosis. Syndecan-2 binds extracellular matrix molecules through its glycosaminoglycan chains. Its cytoplasmic domain is short, but contains two adjacent serine residues that can be phosphorylated, and transfection of renal papillary fibroblasts with a syndecan-2 construct that is truncated at the first serine residue prevents matrix assembly. Furthermore, transfection of renal papillary fibroblasts with syndecan-2 potentiates the increase in fibronectin matrix assembly induced by TGF-1, and syndecan-2 is dramatically upregulated in renal tubulointerstitium in diabetic rats and humans. All of the four members of the syndecan family dimerize and oligomerize, but how this is controlled is not known. Oligomerization of syndecan-4 is needed for biological activity, but this has not been investigated with syndecan-2. The role of syndecan-2 oligomerization in matrix assembly will be monitored. Oligomerization sites will be determined by monitoring hetero-oligomerization of rat and chicken syndecan-2, both in vivo and in vitro, and use of mutated constructs. Syndecan-2 can be phosphorylated, but a role for this in matrix assembly has not been investigated. Regulation of matrix assembly by syndecan-2 phosphorylation will be determined by transfection of constructs where the serine residues are mutated, and by analysis of syndecan-2 phosphorylation status during matrix assembly. Matrix assembly requires an intact cytoskeleton and protein kinase C activity. Interactions of syndecan-2 with other cellular components will be monitored by co-immunoprecipitation and in vitro binding assays. Syndecan-2 potentiation of TGF-1-mediated matrix increase will be studied to determine if growth factor binding is increased, or downstream signaling, using binding assays and TGF-1-dependent promotor activation assays. These studies should determine interaction sites within syndecan-2 for oligomerization and association with other cellular components, and the mechanism underlying the control of matrix assembly by syndecan-4, leading to the identification of new classes of agents for intervention in diabetic fibrosis.
Showing the most recent 10 out of 13 publications
