Abstract. The fibroblast or heparin-binding growth factors (HBGFs) are thought to be modulators of cell growth and migration, angiogenesis, wound repair, neurite extension, and mesoderm induction. A better understanding of the structural basis for the different activities of these proteins should facilitate the development of agonists and antagonists of specific HBGF activities and identification of the signal transduction pathways involved in the mechanisms of action of these growth factors. Chemical modification studies of Harper and Lobb (Harper, J. W., and R. R. Lobb. 1988. Biochemistry. 27:671-678) implicated lysine 132 in HBGF-1 (acidic fibroblast growth factor) as being important to the heparin-binding, receptor-binding, and mitogenic activities of the protein. We changed lysine 132 to a glutamic acid residue by site-directed mutagenesis of the human cDNA and expressed the mutant protein in Escherichia coli to obtain sufficient quantities for functional studies. Replacement of this lysine with glutamic acid reduces the apparent affinity of HBGF-1 for immobilized heparin (elutes at 0.45 M NaC1 vs. 1.1 M NaCI for wild-type). Mitogenic assays established two points: (a) human recombinant I-IBGF-1 is highly dependent on the presence of heparin for optimal mitogenic activity, and (b) the change of lysine 132 to glutamic acid drastically reduces the specific mitogenic activity of HBGF-1. The poor mitogenic activity of the mutant protein does not appear to be due to a reduced affinity for the HBGF receptor. Similarly, the mutant HBGF-1 can stimulate tyrosine kinase activity and induce protooncogene expression. Differences in the biological properties of the wild-type and mutant proteins were observed in transfection studies. Mutant HBGF-1 expression in transfected NIH 3T3 cells did not induce the same transformed phenotype characteristic of cells expressing wild-type HBGF-1. Together these data indicate that different functional properties of I-IBGF-1 may be dissociated at the structural level.
The heparin-binding or fibroblast growth factors (HBGFs) modulate cell growth and migration, angiogenesis, wound repair, neurite extension, and mesoderm induction. Relatively little is known regarding the precise mechanism of action of these growth factors or the structural basis for their action. A better understanding of the structural basis for the different activities of these proteins should lead to the development of agonists and antagonists of specific HBGF activities. In this report, we summarize evidence that indicates that the heparin-binding and mitogenic activities of HBGF-1 can be dissociated from the receptor-binding activities of the growth factor by site-directed mutagenesis of a single lysine residue. Thus, the mutant HBGF-1 has normal receptor-binding activity and is capable of stimulating tyrosine kinase activity and proto-oncogene expression but is not able to elicit a mitogenic response. A similar dissociation of early events such as proto-oncogene expression from the mitogenic response is observed when the human wild-tupe HBGF-1 is used in the absence of added heparin. These results indicate that intracellular sites of action by the growth factor may be required to complete the mitogenic response. Further evidence for this idea is provided by transfection experiments where NIH 3T3 cells are engineered to produce large quantities of wild-type or mutant HBGF-1. Production of wild-type induces a transformed phenotype, whereas over-production of the mutant does not. The majority of both forms of the protein is found in the nuclear fraction of the transfected cells. Additional site-directed mutagenesis of putative nuclear translocation sequences in the wild-type protein do not affect mitogenic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
We previously reported that biomaterials differentially induced macrophages to secrete growth factors that mediate reendothelialization. The present study evaluates the effect of an atherogenic diet on macrophage/biomaterial interactions. Female New Zealand white rabbits were fed an atherogenic diet. Peritoneal macrophages were harvested from these as well as rabbits fed a normal diet and cultured in Minimum Essential Medium with platelet-poor serum. Dacron or polyglactin 910 were added to two of three conditions of both cell groups in passage 2. Conditioned media were collected weekly through week 15. Mitogenicity assays were performed with quiescent mouse embryonal (BALB/c3T3) fibroblasts, atherosclerotic rabbit aortic smooth muscle cells, and murine capillary lung (LE-II) endothelial cells. Mitogenic activity was assayed by scintillation counting of tritiated thymidine incorporation into deoxyribonucleic acid (DNA). Results showed increased mitogenic activity released by macrophages from atherosclerotic rabbits, in the absence of prosthetic material, when assayed against every cell line. In normal diet macrophages, polyglactin 910 stimulated mitogen release for every cell line, and Dacron yielded minimal mitogen release. In lipid diet macrophages polyglactin 910 slightly increased mitogen release for all three cell lines, whereas Dacron resulted in stimulation of DNA synthesis in smooth muscle cells and BALB/c3T3 cells but less DNA synthesis in LE-II cells than in control, no graft material, media. Western blotting demonstrated immunoreactivity to basic fibroblast growth factor in media from normal diet macrophages but only in the presence of polyglactin 910 or Dacron. Radioimmunoassay for platelet-derived growth factor B chain was negative in all groups, and polymerase chain reaction techniques to amplify transforming growth factor-beta messenger ribonucleic was negative. These data demonstrate the effect of in vivo dietary manipulation on macrophage activation as well as the effect of an atherogenic diet in modulating macrophage/biomaterial interactions. Additionally, different biomaterials differentially induce macrophages to release factors that stimulate and inhibit growth.
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