Heparin-binding Growth Factor-1 Research Articles

Enzyme Pretreatment plus Locally Delivered HB-IGF-1 Stimulate Integrative Cartilage Repair In Vitro.

A critical attribute for the long-term success of cartilage defect repair is the strong integration between the repair tissue and the surrounding native tissue. Current approaches utilized by physicians fail to achieve this attribute, leading to eventual relapse of the defect. This article demonstrates the concept of a simple, clinically viable approach for enhancing tissue integration via the combination of a safe, transient enzymatic treatment with a locally delivered, retained growth factor through an in vitro hydrogel/cartilage explant model.

Trypsin Pre-Treatment Combined With Growth Factor Functionalized Self-Assembling Peptide Hydrogel Improves Cartilage Repair in Rabbit Model.

The objective of this study was to improve cartilage repair and integration using self-assembling KLD hydrogel functionalized with platelet-derived growth factor-BB and heparin-binding insulin-like growth factor-1 with associated enzymatic trypsin pre-treatment of the native cartilage. Bilateral osteochondral defects were created at the central portion of the femoral trochlear groove of 48 skeletally mature, white New Zealand rabbits. One limb received a randomly assigned treatment and the contralateral limb served as the control. Treated defects were exposed to trypsin for 2 min and filled with self-assembling KLD hydrogel only, or associated to growth factors. All control limbs received KLD hydrogel alone or received only trypsin but not hydrogel. Ninety days post-defect creation, the rabbits were euthanized and magnetic resonance imaging, radiography, macroscopic evaluation, histology, and immunohistochemistry of the joint and repaired tissue were performed. Mixed model analyses of variance were utilized to assess the outcome parameters and individual comparisons were performed using Least Square Means procedure and differences with p-value < 0.05 were considered significant. Trypsin enzymatic pre-treatment improved cellular morphology, cluster formation and subchondral bone reconstitution. Platelet-derived growth factor-BB improved subchondral bone healing and basal integration. Heparin-binding insulin-like growth factor-1 associated with platelet-derived growth factor improved tissue and cell morphology. The authors conclude that self-assembling KLD hydrogel functionalized with platelet-derived growth factor and heparin-binding insulin-like growth factor-1 with associated enzymatic pre-treatment of the native cartilage with trypsin resulted in an improvement on the cartilage repair process. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2307-2315, 2019.

Growth Factor-Mediated Migration of Bone Marrow Progenitor Cells for Accelerated Scaffold Recruitment.

Tissue engineering approaches using growth factor-functionalized acellular scaffolds to support and guide repair driven by endogenous cells are thought to require a careful balance between cell recruitment and growth factor release kinetics. The objective of this study was to identify a growth factor combination that accelerates progenitor cell migration into self-assembling peptide hydrogels in the context of cartilage defect repair. A novel 3D gel-to-gel migration assay enabled quantification of the chemotactic impact of platelet-derived growth factor-BB (PDGF-BB), heparin-binding insulin-like growth factor-1 (HB-IGF-1), and transforming growth factor-β1 (TGF-β1) on progenitor cells derived from subchondral bovine trabecular bone (bone-marrow progenitor cells, BM-PCs) encapsulated in the peptide hydrogel [KLDL]3. Only the combination of PDGF-BB and TGF-β1 stimulated significant migration of BM-PCs over a 4-day period, measured by confocal microscopy. Both PDGF-BB and TGF-β1 were slowly released from the gel, as measured using their (125)I-labeled forms, and they remained significantly present in the gel at 4 days. In the context of augmenting microfracture surgery for cartilage repair, our strategy of delivering chemotactic and proanabolic growth factors in KLD may provide the necessary local stimulus to help increase defect cellularity, providing more cells to generate repair tissue.

Identification of a 79-kDa Heparin-binding Fibroblast Growth Factor (FGF) Receptor in Rat Hepatocytes and Its Correlation with the Different Growth Responses to FGF-1 between Hepatocyte Subpopulations

We reported previously that the potency of heparin-binding fibroblast growth factor-1 (FGF-1) as a mitogen for rat hepatocytes in primary culture is as high as that of epidermal growth factor (EGF) and hepatocyte growth factor. To gain insight into the pathophysiological significance of FGF-1 in hepatocyte growth, we analyzed the cooperative mitogenicity of FGF-1 and EGF. Results from a nuclear labeling assay using [3H]thymidine suggest that most hepatocytes in primary culture consist of two cell populations that differ in response to FGF-1; one is an FGF-1-responsive cell population, and the other is an EGF-responsive (but not FGF-1-responsive) cell population. On the other hand, autoradiographic analysis of 125I-FGF-1 binding demonstrated that high affinity FGF receptors were homogeneously distributed on the surface of all hepatocytes. Cross-linking 125I-FGF-1 to the nonstimulated hepatocyte surface indicated that the high affinity FGF receptors comprise two FGF receptors that differ in molecular mass (128 and 79 kDa). Furthermore, the 79-kDa receptor was preferentially down-regulated when the hepatocytes were stimulated with EGF or hepatocyte growth factor. These data suggest that the abundant expression of the 79-kDa FGF receptor on some populations of hepatocytes is involved in their lack of response to FGF-1. The 128- and 79-kDa FGF receptors were assigned as FGFR2 using an antibody specific to the ectodomain of FGFR2, whereas the 79-kDa receptor was not reactive to the antibody against the carboxyl terminus of FGFR2. This 79-kDa FGF receptor was not tyrosine-phosphorylated in response to FGF-1 stimulation, while the 128-kDa FGF receptor was recognized by anti-phosphotyrosine antibody under the same conditions. Also, the heterodimer of 79- and 128-kDa FGF receptors was less tyrosine-phosphorylated than the homodimer of 128-kDa FGF receptors. These data suggest that the 79-kDa FGF receptor inhibits the function of the 128-kDa FGF receptor through their heterodimerization. Thus, we surmise that the difference in response to FGF-1 between the cell populations of normal rat hepatocytes was caused by the different levels of the 79-kDa FGF receptor in each cell population.

Monoclonal Antibodies against Heparin-Binding Growth Facto 1: Neutralization of Biological Activity and Recognition of Specific Amino Acid Sequence

A panel of three monoclonal antibodies against heparin-binding growth factor-1 (HBGF-1) was obtained. These antibodies, Ab-47α, Ab-15, Ab-29, were able to recognize HBGF-1 but not HBGF-2. One of the antibodies, Ab-47α, was identified as a HBGF-1 neutralizing antibody on the basis of its ability to inhibit the binding of [125I]HBGF-1 to receptors on HepG-2 cells and the proliferation of fetal bovine heart endothelial cells induced by HBGF-1. Ab-15 reacted with truncated HBGF-1(Mr= 16,000), intact HBGF-1( Mr= 18,000) and mutant HBGF-1U which lacks a putative nuclear translocation sequence (amino acid residues 21 to 27 of HBGF-1). Ab-29 reacted with only truncated HBGF-1 and was thought to recognize the putative nuclear translocation sequence of HBGF-1. The three monoclonal antibodies did not inhibit the binding of [125I]HBGF-1 to heparin. These data indicate that each monoclonal antibody recognizes a distinct epitope of HBGF-1 and identifies them as useful reagents for evaluating functional domains and biological roles of HBGF-1.

Acidic fibroblast growth factor gene 5' non-coding exon and flanking region from hamster DDT1 cells: identification of the promoter region and transcriptional regulation by testosterone and aFGF protein.

Selected clones of Syrian hamster DDT1-MF2 cells are responsive to testosterone for growth. Heparin binding growth factor 1 (HBGF-1) or acidic fibroblast growth factor (aFGF) can replace testosterone (T) in the stimulation of growth in these cells. This phenomena is correlated with testosterone's ability to elevate aFGF mRNA two- to threefold in DDT1 cells. To better understand the possible mechanisms of regulation of aFGF mRNA by steroids and other growth factors, we isolated the aFGF 5' non-coding exon and its flanking region from a EMBL3 DDT1 genomic library, using a 5' non-coding exon 69 bp DDT1 aFGF cDNA probe. Clones spanning 30 kb of genomic DNA were isolated. After restriction mapping and DNA sequence analysis, the clones were shown to contain all of the 5' non-coding exon included in the cDNA and approximately 10 kb of 5' flanking region. RNase protection and primer extension assays confirmed that the 5' non-coding exon is included in the DDT1 aFGF mRNA and that a major transcription start site is approximately 136 bp upstream of the 5' non-coding splice junction of this exon. The 5' flanking region DNA was inserted into pBLCAT3 reporter gene and transfected into DDT1 cells. Chloramphenicol acetyltransferase (CAT) assays demonstrated that there are promoter elements in the -1645/-392 and -392/+131 regions of the aFGF gene in the context of DDT1 cells. NIH 3T3 cells, on the other hand, show no CAT activity with these aFGF-CAT plasmids. CAT assays also demonstrated that addition of testosterone (T) or aFGF to DDT1 cells increased CAT activity threefold. This activity was mapped to -1645 to -4 bp region of this DDT1 aFGF gene promoter.

Purification and characterization of fibroblast growth factors from the opossum, Monodelphis domestica, brain.

An extract from the brain of the opossum Monodelphis domestica was fractionated by heparin affinity chromatography. A major peak of mitogenic activity (heparin binding growth factor 2, HBGF-2) eluted from heparin-Sepharose between 1.7 and 2.0 M NaCl. Antisera specific for bovine bFGF detected four polypeptides of 17.5-23 kDa in opossum brain HBGF-2 preparations. Opossum brain heparin binding growth factor 1 (HBGF-1), a minor peak of activity, eluted from heparin-Sepharose at 1.1 NaCl and contained a 16.2 kDa protein that cross-reacted with antiserum against bovine aFGF.

Identification of a heparin-binding growth factor-1 nuclear translocation sequence by deletion mutation analysis.

We have shown previously that a deletion mutant of human heparin-binding growth factor (HBGF)-1, HBGF-1U, lacking the sequence Asn-Tyr-Lys-Lys-Pro-Lys-Leu is capable of initiating c-fos mRNA expression and polypeptide phosphorylation on tyrosine residues at concentrations that do not induce either DNA synthesis or cell proliferation (1). The fact that addition of the nuclear translocation signal from the yeast histone 2B protein to the HBGF-1U mutant caused reconstitution of the biological activity of HBGF-1 indicated that nuclear translocation may be an important component of the mitogenic signal induced by HBGF-1. In order to examine the nuclear translocation potential of HBGF-1 alpha, the deletion mutant HBGF-1U, and the yeast histone 2B-HBGF-1 chimera, HBGF-1U2, we expressed these forms of HBGF-1 in murine endothelial cells. Western blot and two-dimensional Western blot analysis of cytosol and nuclei demonstrate that although the three forms of HBGF-1 are readily detectable in the cytosol of the individual transfectants, HBGF-1 alpha and HBGF-1U2 but not HBGF-1U was detected in the nucleus. Furthermore, murine endothelial cells expressing HBGF-1 alpha and HBGF-1U2 exhibited an atypical cellular phenotype in vitro that was absent in the HBGF-1U transfectants. These data suggest that HBGF-1 contains a functional nuclear translocation sequence that may be responsible for the initiation of DNA synthesis, and these data further correlate the presence of the nuclear translocation sequence with an abnormal endothelial cell phenotype in vitro.

Molecular Biology of the Coronary Vascular and Myocardial Responses to Ischemia

To understand the complex mechanism(s) involved in molecular responses to ischemia, we developed two experimental models in pigs. In a "stunning" model of repetitive ischemia and reperfusion, we studied the mRNA expression of immediate early genes like c-fos, c-myc and heat shock protein-70 (HSP-70). Myocardial stunning was achieved by two cycles of 10-min left anterior descending coronary artery (LAD) occlusion and 30 min reperfusion. We observed several-fold enhanced expression of c-fos and HSP-70 mRNA in the stunned myocardium as compared with the control, whereas c-myc mRNA levels remained almost unchanged. In the second model, we examined the expression of the peptide mitogens heparin-binding growth factor 1 (HBGF-1) and transforming growth factor beta 1 (TGF-beta 1) after a chronic coronary artery occlusion leading to myocardial collateralization. Progredient stenosis of the circumflex coronary artery was induced by implanting a hygroscopic ameroid constrictor ring around it and occlusion was verified by in vivo angiography. Using polymerase chain reaction (PCR) and Northern hybridization techniques, we observed significantly enhanced expression of HBGF-1 and TGF-beta 1 in collateralized myocardium as compared with normal. In situ techniques revealed the localization of HBGF-1 transcripts in the blood vessel wall, and TGF-beta 1 in cardiac myocytes and Purkinje cells. Our results clearly indicate that myocardial stunning stimulates the expression of transcription factors which might be involved in regulation of certain growth factors like HBGF-1 and TGF-beta 1 which may play a significant role in the development of a collateral circulation.

Cyclooxygenase gene expression is down-regulated by heparin-binding (acidic fibroblast) growth factor-1 in human endothelial cells.

The expression of cyclooxygenase (EC 1.14.99.1, Cox), a rate-limiting enzyme in the biosynthesis of inflammatory prostanoids, is regulated by growth factors and cytokines. We have shown previously that the cytokine interleukin-1, an inhibitor of endothelial growth in vitro and stimulator of prostacyclin production, induces the expression of the 3-kilobase Cox transcript in cultured human umbilical vein endothelial cells (HUVEC). In contrast, the endothelial cell mitogen, heparin-binding (acidic fibroblast) growth factor-1 (HBGF-1) inhibits the synthesis of prostacyclin in HUVEC. In this report, we describe the effect of HBGF-1 on Cox mRNA expression in HUVEC. Cells cultured in the presence of HBGF-1 express diminished Cox mRNA levels whereas quiescent cells maintained in serum expressed a 7-fold higher level of the transcript for Cox. Concomitantly, the level of the Cox translation product and prostacyclin synthesis are also reduced by HBGF-1. Further, HBGF-1, in the presence of heparin, down-regulates the levels of the Cox transcript in a dose- and time-dependent manner. The onset of action of HBGF-1 is slow, requiring up to 24 h to depress the level of Cox mRNA. Lastly, the effective dose of HBGF-1 to depress Cox mRNA levels is similar to that required for mitogenesis, suggesting that cell proliferation may be required for the reduction of Cox expression in vitro. Thus, Cox may belong to a class of genes that are reversibly down-regulated during periods of endothelial cell proliferation.

Differential expression in Escherichia coli of the α and β forms of heparin-binding acidic fibroblast growth factor-1: potential role of RNA secondary structure

Synthetic DNA fragments encoding the entire open-reading frame of human heparin-binding growth factor-1 (HBGF-1 beta) and its NH2-terminal truncated form (HBGF-1 alpha) were constructed. When both constructs were expressed in Escherichia coli under control of the trp-lac promoter, biologically active HBGF-1 alpha, but not HBGF-1 beta was produced in high yield. However, high level expression of HBGF-1 beta was obtained using the T7 polymerase expression vector. Computer analysis of HBGF-1 beta predicts the potential for the formation of exaggerated RNA secondary structure near the translation initiation codon and this could be implicated in contributing to the poor translation of HBGF-1 beta under the trp-lac promoter.

Tissue- and development-specific expression of HBGF-1 mRNA

The gene for heparin-binding growth factor-1 (HBGF-1) encodes a 15.5-18 kDa polypeptide that affects the proliferation and differentiation of a broad range of mammalian cells and is widely distributed among normal adult tissues. In this study, we show that normal tissues of the adult rat express HBGF-1 transcripts in one of three patterns: a 4.4 kb mRNA was the predominant HBGF-1 transcript in brain, heart and lung; a 1.4 kb mRNA was the predominant transcript in the liver; approximately equal levels of the 1.4 and 4.4 kb mRNAs were found in the kidney. HBGF-1 expression was localized in two tissues: central nervous system expression of HBGF-1 was significantly higher in the brain stem compared to the cerebrum and cerebellum; renal expression of HBGF-1 was significantly higher in the medulla compared to the cortex. Analysis of the postnatal changes in HBGF-1 expression using the newborn rat kidney revealed that the level of HBGF-1 mRNA is low at birth and does not rise to adult levels until the seventh postnatal day. These findings demonstrate that HBGF-1 expression is specific for tissue type and stage of development.

Mechanism for diminished tissue factor expression by endothelial cells cultured with heparin binding growth factor-1 and heparin

We have extended our earlier observation that growing primary cultures of human umbilical vein endothelial cells (HUVEC) with heparin binding growth factor 1 (HBGF-1) 20 micrograms/mL and heparin 12 U/mL inhibits expression of tissue factor (TF) activity on HUVC monolayers perturbed with thrombin. TF activity was measured as the ability of monolayers or cell lysates to support FVIIa-catalyzed activation peptide release from 3H-FX. TF antigen in HUVEC extracts was measured in an enzyme-linked immunosorbent assay (ELISA) that uses a double-antibody sandwich technique with rabbit and goat antibodies to human TF. TF-mRNA was measured by Northern blot hybridization with a 32P-TF cDNA probe. Cells growth with HBGF-1/heparin had both decreased surface and total TF activity as compared with HUVEC from the same endothelial cell pool grown without HBGF-1/heparin. Means +/- SD for TF antigen for four primary cultures were 4.4 +/- 0.9 ng/10(6) cells without HBGF-1/heparin and 0.6 +/- 0.3 ng/10(6) cells with HBGF-1/heparin. TF mRNA 4 hours after incubation with thrombin of HUVEC grown without HBGF-1/heparin was about sevenfold higher than TF mRNA of HUVEC grown with HBGF- 1/heparin. These data establish that growing primary cultures of HUVEC with HBGF-1/heparin impairs their ability to synthesize TF apoprotein after perturbation. This may be part of a generalized response of endothelial cells to HBGF-1/heparin facilitating migration during angiogenesis.

Transformed phenotype conferred to NIH/3T3 cells by ectopic expression of heparin-binding growth factor 1/acidic fibroblast growth factor

Heparin-binding growth factor 1 (HBGF-1), also known as acidic fibroblast growth factor, is a potent mitogen and angiogenic factor found in tissues such as brain, kidney and heart. The genomic and cDNA sequences indicate that HBGF-1 does not have a typical signal peptide sequence. HBGF-1 was shown to be localized to the extracellular matrix of cardiac myocytes, but the mechanism of secretion is not presently known. We have cloned the HBGF-1 cDNA which allowed us to directly test the biological activity, mechanism of secretion and transforming potential of the recombinant protein. A previous report showed that the truncated HBGF-1 confers partial transformed phenotype to the recipient fibroblasts. However, expression of full-length HBGF-1 has not been reported. The HBGF-1 coding sequence was cloned into the retroviral expression vector, SVX, and transfected into NIH/3T3 cells. Transfectants expressing full-length HBGF-1 protein at high levels form foci and grow to a higher cell density than the parental NIH/3T3 cells. Western blotting analysis showed that the recombinant HBGF-1 is a unique band of approximately 20 kDa and can be detected in the cell homogenate but not in the conditioned medium. NIH/3T3 cells were conferred anchorage independence when HBGF-1 was provided exogenously. We showed the transformed cells are capable of growing on soft agar even in the absence of exogenously-provided HBGF-1. Transfected cells expressing HBGF-1 also induced tumor formation when injected into nude mice. Thus NIH/3T3 cells acquired a full spectrum of transformed phenotype when full length HBGF-1 was expressed at high levels.

Liver contains heparin-binding growth factors as the major growth factor for cultured fibroblasts

The presence of heparin-binding growth factors in liver was investigated by measuring the DNA synthesis stimulatory activity of liver extracts using quiescent fibroblasts as target cells. It was found that cytosolic fractions of mouse, rat and human liver, as well as isolated rat hepatocytes, contain a large amount of growth stimulatory activity. Most liver cytosolic activity is due to heparin-binding growth factors, because > 90% of the activity bound to a heparin affinity column in the presence of 0.8 mol/L NaCl, and was quantitatively eluted with 2 mol/L NaCl. Purification of these factors from both mouse and rat liver indicated the presence of both heparin-binding growth factor-1 and 2 in liver extracts. The level of the heparin-binding growth factors, as estimated from the biological activity, is approximately 1 μg/gm mouse liver and 0.1 μg/gm rat and human liver. Heparin-binding growth factor-1–like factors were 10 times as abundant as heparin-binding growth factor-2–like factors. These data indicate that the cytosolic fractions of mouse, rat and human liver contain heparin-binding growth factors as the primary growth factor for fibroblasts, and heparin binding growth factor-1–like molecules account for most of the cytosolic activity in both mouse and rat liver. (HEPATOLOGY 1991;13:6–14).

Liver contains heparin-binding growth factors as the major growth factor for cultured fibroblasts

The presence of heparin-binding growth factors in liver was investigated by measuring the DNA synthesis stimulatory activity of liver extracts using quiescent fibroblasts as target cells. It was found that cytosolic fractions of mouse, rat and human liver, as well as isolated rat hepatocytes, contain a large amount of growth stimulatory activity. Most liver cytosolic activity is due to heparin-binding growth factors, because greater than 90% of the activity bound to a heparin affinity column in the presence of 0.8 mol/L NaCl, and was quantitatively eluted with 2 mol/L NaCl. Purification of these factors from both mouse and rat liver indicated the presence of both heparin-binding growth factor-1 and 2 in liver extracts. The level of the heparin-binding growth factors, as estimated from the biological activity, is approximately 1 microgram/gm mouse liver and 0.1 microgram/gm rat and human liver. Heparin-binding growth factor-1-like factors were 10 times as abundant as heparin-binding growth factor-2-like factors. These data indicate that the cytosolic fractions of mouse, rat and human liver contain heparin-binding growth factors as the primary growth factor for fibroblasts, and heparin binding growth factor-1-like molecules account for most of the cytosolic activity in both mouse and rat liver.

TGF‐β inhibits the platelet‐derived growth factor‐induced formation of inositol trisphosphate in MG‐63 human osteosarcoma cells

Epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and heparin-binding growth factor-1 (HBGF-1) stimulated the proliferation of a variant of the human osteosarcoma cell line, MG-63-LS (LS = low serum). Transforming growth factor beta (TGF-beta) completely inhibited cell growth in basal medium supplemented with 2% fetal calf serum (FCS), blocked PDGF- and EGF-stimulated cell proliferation, and modulated that of HBGF-1. PDGF, but not EGF or HBGF-1, activated the inositol trisphosphate/diacylglycerol (IP3/DAG) second message system in a dose-dependent manner. EGF inhibited phosphoinositol lipid turnover and HBGF-1 and TGF-beta stimulated phosphatidylinositol hydrolysis to produce inositol phosphate (IP) but not IP3. Preincubation of quiescent cells with TGF-beta for 30-40 minutes prior to the addition of PDGF resulted in an inhibition of PDGF-induced production of IP3. This suggested that TGF-beta was an indirect inhibitor and blocked PDGF-stimulated cell growth in part by interfering with the generation of the second messenger, IP3.

Heparin-binding growth factor-1 modulation of plasminogen activator inhibitor-1 expression. Interaction with cAMP and protein kinase C-mediated pathways.

Plasminogen activator inhibitor-1 (PAI-1) inhibits the tissue plasminogen activator (tPA) and urokinase activation of plasminogen to plasmin, a protease of trypsin-like specificity which is involved in a number of processes, including fibrinolysis, matrix degradation and angiogenesis. Both phorbol esters and cAMP elevating compounds have been shown to modulate PAI-1 and tPA expression in endothelial cell culture. HBGF-1 (previously designated endothelial cell growth factor) stimulates endothelial cell growth in vitro and is angiogenic in vivo. We have reported that removal of HBGF-1 from human umbilical vein endothelial cell (HUVEC) media results in an approximately 5-fold increase in PAI-1 mRNA levels and in PAI-1 protein secreted into the media by 20 h. Here we report the effects of HBGF-1 on the phorbol ester and cAMP modulation of HUVEC PAI-1 expression. The phorbol ester PMA induced an approximate 5-fold increase in PAI-1 mRNA levels at 4 h, which returned to base line by 20 h, with or without HBGF-1 present in the media. This increase in PAI-1 mRNA levels was mediated by an increase in PAI-1 gene transcription and was abated in the presence of cycloheximide. Treatment of cells with the adenylate cyclase activator forskolin or the phosphodiesterase inhibitor HL 725, in the presence of HBGF-1 or immediately after its withdrawal, decreased PAI-1 mRNA levels and protein secreted into the conditioned media by 20 h. However, forskolin or HL 725 addition had little or no effect on PAI-1 mRNA when added 20 h after HBGF-1 withdrawal. Both the PMA and HBGF-1 modulation of PAI-1 were abolished by treatment with the protein kinase inhibitor H-7. Treatment of HUVEC with HBGF-1 had no acute effect on intracellular inositol phosphate hydrolysis or cAMP levels. Further studies on intracellular pathways involved in HBGF-1 modulation of PAI-1 will enhance our understanding of the role these factors play in cellular proliferation and angiogenesis.

Possible dissociation of the heparin-binding and mitogenic activities of heparin-binding (acidic fibroblast) growth factor-1 from its receptor-binding activities by site-directed mutagenesis of a single lysine residue.

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 NaCl vs. 1.1 M NaCl for wild-type). Mitogenic assays established two points: (a) human recombinant HBGF-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 HBGF-1 may be dissociated at the structural level.

Recovery of Mitogenic Activity of a Growth Factor Mutant with a Nuclear Translocation Sequence

Heparin-binding growth factor-1 (HBGF-1) is an angiogenic polypeptide mitogen for mesoderm- and neuroectoderm-derived cells in vitro and remains biologically active after truncation of the amino-terminal domain (HBGF-1 alpha) of the HBGF-1 beta precursor. Polymerase chain reaction mutagenesis and prokaryotic expression systems were used to prepare a mutant of HBGF-1 alpha lacking a putative nuclear translocation sequence (amino acid residues 21 to 27; HBGF-1U). Although HBGF-1U retains its ability to bind to heparin, HBGF-1U fails to induce DNA synthesis and cell proliferation at concentrations sufficient to induce intracellular receptor-mediated tyrosine phosphorylation and c-fos expression. Attachment of the nuclear translocation sequence from yeast histone 2B at the amino terminus of HBGF-1U yields a chimeric polypeptide (HBGF-1U2) with mitogenic activity in vitro and indicates that nuclear translocation is important for this biological response.