The prevention of thrombosis is an important function of the endothelium in vivo and the maintenance of endothelial integrity is a presumed function of the heparin-binding fibroblast growth factors (HBGFs). Previous efforts from this laboratory have defined the identity, function and structure of HBGF-1, an angiogenic prototype of the HBGF family and have also elucidated similar structure-function relationships for the HBGF-1 receptor. A unique feature of the HBGF prototypes is the lack of a signal sequence for secretion and while the prior application involved studies directed toward the function of HBGF-1 receptor, this feature has led this laboratory to question whether HBGF-1 can function as an intracellular polypeptide. Indeed, recent evidence from this laboratory suggests that HBGF-1 and its structural homolog IL-1alpha can function as intracellular regulators of cell division in vitro and HBGF-1 may utilize nuclear translocation as a component of its ability to induce cell proliferation. Thus, a specific aim of this competitive renewal application is to define the minimum structure for nuclear localization in order to understand how HBGF-1 is partitioned between cytosol and the nucleus. The identification of domains within the structure of HBGF-1 and the elucidation of the structure of putative intracellular binding proteins that may participate in the partitioning of HBGF-1 between cytosol and nucleus are among these goals. These studies will employ mutagenesis of the nuclear translocation sequence and HBGF-1 ligand affinity cloning. Conventional cloning strategies are also discussed and will employ ligand affinity chromatography for the purification of cytosol- and nuclei-derived polypeptides that are able to associate with HBGF-1. Likewise, HBGF-1 is also able to associate with DNA, is phosphorylated on serine residues and is able to form stable homo and heterodimers, and while it is not known whether these interactions are indeed significant, we propose that they are interesting, reproducible and worthy of further study. Thus, another specific aim of this application is to define whether the interaction between HBGF-1 and DNA involves specific nucleotide sequences, possibly mediate through unknown cytosolic or nuclear polypeptides, or whether this association involves the ability of HBGF-1 to recognize the phosphodiester backbone. If the latter interaction is accurate, we propose to take advantage of the non-specific interaction between HBGF-1 and DNA for the delivery of IL-1alpha antisense oligomers during adjuvant-induced inflammation in the rat. If successful, this approach may enable us to study, define and regulate angiogenic phenomena during experimental solid tumor growth, atherogenesis, rheumatoid arthritis and other pathophysiologic situations in vivo.
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