Endothelial cell acidic fibroblast growth factor binding proteins

Abstract

This thesis has focused on endothelial cell proteins which interact with fibroblast growth factor-1 (FGF-1). Endothelial cell extracellular matrix (ECM) heparan sulfate proteoglycans (HSPGs) which interact with FGF-1 play an important role in controlling cell growth. We have investigated the ability of ECM HSPG{dollar}\sp{lcub}\rm P{rcub}{dollar} (potentiator) isolated from different passages to augment the FGF-1 mitogenic activity. HUVEC was demonstrated to produce ECM HSPGs that are potent endothelial cell growth potentiator (HSPG{dollar}\sp{lcub}\rm P{rcub}{dollar}) and inhibitor (HSPG{dollar}\sp{lcub}\rm I{rcub}{dollar}). HSPG{dollar}\sp{lcub}\rm P{rcub}{dollar} extracted from the ECM of cultured endothelial cells bind FGF-1 and potentiate the mitogenic activity of FGF-1. Deposition of HSPG{dollar}\sp{lcub}\rm P{rcub}{dollar} in the ECM was induced by heparin and FGF-1. The late passaged endothelial cells produce decreased amounts of ECM HSPG{dollar}\sp{lcub}\rm P{rcub}{dollar}. In addition, the HSPG{dollar}\sp{lcub}\rm P{rcub}{dollar} from late passaged cells were less sulfated and exhibited a decreased ability to augment FGF-1 mitogenic activity. The ECM HSPG{dollar}\sp{lcub}\rm P{rcub}{dollar} from late passaged cells gradually lost the ability to augment FGF-1 mitogenic activity.;In an attempt to understand the nuclear events associated with FGF-1 the cytosolic and nuclear proteins of HUVEC which interact with FGF-1 were isolated and identified.;We identified four (68, 78, 90 and 120kD) cytosolic proteins which interact with FGF-1. Partial N-terminal amino acid sequence of the 68kD protein demonstrated that it was related to human calreticulin, a calcium binding protein. In addition, recombinant skeletal muscle calreticulin, a calcium binding protein, was found not to bind a mutant FGF-1 in which the NLS sequence (-NYKKPKL-) was deleted. Also calreticulin was shown to enhance the accumulation of FGF-1, NLS peptide, and SV 40 large T antigen NLS peptide fluorescent conjugate in the nuclei of digitonin permeabilized HUVEC and NRK 52E cells.;We isolated two nuclear proteins, one 52kD and the other 63kD, which interact with FGF-1. The 63kD protein, which was isolated utilizing HSPG{dollar}\sp{lcub}\rm P{rcub}{dollar}-chromatography, interacted with a peptide containing the putative FGF-1 NLS sequence (CGGGNYKKPKL) and to a lesser degree to a synthesized NLS mutant (CGGGNYTKPKL). In an in vitro nuclear binding assay, saturable binding of {dollar}\sp{lcub}125{rcub}{dollar}I-HSA-NLS and {dollar}\sp{lcub}125{rcub}{dollar}I-FGF-1 was demonstrated. Less binding of {dollar}\sp{lcub}125{rcub}{dollar}I-HSA-NLS mutant and {dollar}\sp{lcub}125{rcub}{dollar}I-FGF-1U (mutant, NLS-NYKKPKL-deleted) to isolated HUVEC nuclei was observed. The p63 HUVEC nuclear envelope protein inhibited nuclear binding of both the {dollar}\sp{lcub}125{rcub}{dollar}I-HSA-NLS and {dollar}\sp{lcub}125{rcub}{dollar}I-FGF-1 to isolated nuclei. Using an in vitro nuclear import assay the import of FGF-1 and fluorescent conjugates of the NLS peptides into the nucleus, were found to require ATP and to be temperature dependent. Also, the NLS of FGF-1 was required for binding and translocation of FGF-1 into the nucleus. In addition the 63kD nuclear envelope protein inhibited the import of FGF-1 into the nucleus. The 52kD protein on the other hand was identified to be related to calreticulin, based on the partial N-terminal amino acid sequence. Thus we have identified and partially characterized endothelial cell proteins which interact with FGF-1 and modulate nuclear binding and transport of FGF-1 into the nucleus. (Abstract shortened by UMI.).