Heparin potentiates the action of acidic fibroblast growth factor by prolonging its biological half-life

The mechanism(s) by which heparin influences the biological activities of acidic and basic fibroblast growth factors (aFGF and bFGF) is not completely understood. One mechanism by which heparin could alter the biological activities of aFGF and bFGF is by altering their biological half-lives. We investigated the possibility that heparin potentiates aFGF-induced neurite outgrowth from PC12 cells by prolonging its biological half-life. Under conditions where heparin potentiated aFGF-induced neurite outgrowth, we observed that heparin increased the biological half-life of aFGF from 7 to 39 hr. We determined that greater than 25 hr of exposure to active aFGF was required for induction of neurite outgrowth. If aFGF activity was maintained for greater than 25 hr by periodic readdition of factor, heparin no longer potentiated aFGF-induced neurite outgrowth. These observations strongly suggest that heparin potentiates the activity of aFGF by prolonging its biological half-life. The protease inhibitors hirudin, leupeptin, and pepstatin A did not potentiate aFGF-induced neurite outgrowth, indicating that proteases inhibited by these inhibitors are not responsible for the loss of aFGF activity that we observed. However, aprotinin potentiated aFGF neurite-promoting activity approximately sevenfold, indicating that proteases that are inhibited by aprotinin are at least partially responsible for aFGF inactivation. These observations suggest that heparin regulates the activity of aFGF by regulating its proteolytic degradation, thereby regulating its biological half-life.     

Growth-stimulatory action of plasma membrane-associated growth factor. A synergistic effect with platelet-poor plasma

Growth factor activity was partially purified from mouse liver plasma membranes and its growth-stimulatory action on cultured mouse fibroblasts was studied. The plasma membrane-associated growth factor (PMGF) was unable to support the proliferation of mouse fibroblasts in monolayer when added as the sole source of growth factor. However, it stimulated the growth of fibroblasts in the presence of CM-Sephadex-treated human platelet-poor plasma (h-CMP) which by itself is not growth-stimulatory. The stimulation of DNA synthesis in quiescent fibroblasts was also observed upon the addition of PMGF and h-CMP. Under the same conditions, both platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) showed the same effect as did PMGF. The synergistic action of h-CMP with PMGF on quiescent cells was partially reproduced by insulin at microgram quantities or by insulin-like growth factor I(IGF-I) at nanogram quantities. Thus, the data presented here indicates that the action of PMGF is similar to that of the family of growth factors termed 'competence factor', and distinct from that of plasma growth factors termed 'progression factor'.     

Purification of melanoma growth stimulatory activity

The Hs0294 human malignant melanoma cell line produces a monolayer mitogen that stimulates the serum free growth of low-density cultures of Hs0294 cells. This report describes the purification of that mitogen, termed MGSA for melanoma growth stimulatory activity, from serum-free conditioned medium from the Hs0294 cultures. MGSA has been purified from acetic acid extracts of lyophilized conditioned medium by gel filtration, reverse-phase high-pressure liquid chromatography (RP-HPLC), and preparative electrophoresis, resulting in a greater than 400,000-fold purification. MGSA bioactivity resides in acid- and heat-stable polypeptides of high and low molecular weight (24-28 kd and less than 14-16 kd). However, the majority of the activity is reproducibly associated with the approximately 16-kd moiety eluting from RP-HPLC at approximately 35% acetonitrile. Reduction with dithiothreitol or B-mercaptoethanol results in a loss of biological activity but does not convert the 24-28-kd moieties to the less than 14-16-kd forms of MGSA. 125I-MGSA that has been purified by preparative electrophoresis (16 kd) specifically binds to Hs0294 melanoma cells and retains 100% of the growth-stimulatory activity. The 16-kd MGSA stimulates the proliferation of Hs0294 cells at concentrations of 0.3-30 pM. The electrophoretic mobility of MGSA is also unaltered by the preparative electrophoresis procedure, further demonstrating that this procedure does not alter the biochemical integrity of the growth factor. Purified MGSA does not enable anchorage-independent growth of normal rat kidney (NRK) cells and is therefore different from the previously described transforming growth factors. The amino acid composition of MGSA differs from that of other previously described growth factors. These data demonstrate that MGSA represents a separate class of growth factors with biological and biochemical properties different from other growth factors.     

Vanadate potentiates the glycogenic action of insulin-like growth factors on isolated diaphragm.

Na3VO4 (6.5 mumol/100 g rat weight), co-injected with a trace amount of [14C]glucose, increased within 15 min the incorporation of radiolabel in diaphragmal glycogen. After 2 h the vanadate-induced increases were 12-fold in the diaphragm and 7-8-fold in heart and liver. In contrast, when added to isolated diaphragms for up to 1 h, vanadate (0.1-5 mM) had no effect on the synthesis of glycogen from 5 mM glucose. In search of a putative mediator of vanadate's action in vivo, insulin and the insulin-like growth factors (IGFs) were considered. Their plasma concentration was not affected by vanadate treatment. In isolated diaphragms, 1 mM vanadate did not potentiate insulin-induced glycogen synthesis, but it caused a several-fold increase in glycogen synthesis in the presence of concentrations of IGF-I which, alone, had no effect. A similar synergism occurred between vanadate and IGF-II. We propose that the glycogenic action of vanadate in vivo, at least in some tissues, involves a potentiation of the action of IGF-I.     

Bezafibrate decreases growth stimulatory action of the sphingomyelin signaling pathway in regenerating rat liver

Liver regeneration after partial hepatectomy (PH) is achieved through proliferation of hepatocytes and non-parenchymal cells. The nuclear peroxisome proliferator-activated receptor alpha (PPARalpha) is involved in regulation of lipid metabolism and proliferation of hepatic cells. The sphingomyelin signal transduction pathway is involved in the regulation of the cell cycle in eukaryotic organisms. Sphingosine-1-phosphate (S1P) and ceramide (CER)-- the intermediates of the pathway--are known to stimulate and to inhibit cellular proliferation. The aim of the present study was to investigate the effect of PPARalpha activation by bezafibrate on the sphingomyelin signaling pathway during the first 24h of liver regeneration after PH in the rat. The content of sphingomyelin, ceramide, sphingosine, sphinganine, sphingosine-1-phosphate and the activity of sphingomyelinases and ceramidases were determined at various time points after PH. It has been found that the activity of neutral Mg(2+)-dependent sphingomyelinase (nSMase) increased, whereas the activity of acidic sphingomyelinase (aSMase) decreased in the regenerating liver. Activation of PPARalpha by bezafibrate lower the activity of nSMase and increased the activity of aSMase in the regenerating rat liver. The content of ceramide was higher in bezafibrate-treated rats, whereas the content of sphingosine-1-phosphate was markedly lower as compared to the untreated rats. Therefore, it is concluded that activation of PPARalpha by bezafibrate decreases the growth-stimulatory activity of the sphingomyelin pathway in regenerating rat liver.     

Heparin affin regulatory peptide/pleiotrophin mediates fibroblast growth factor 2 stimulatory effects on human prostate cancer cells

Fibroblast growth factor 2 (FGF2) is a pleiotropic growth factor that has been implicated in prostate cancer formation and progression. In the present study we found that exogenous FGF2 significantly increased human prostate cancer LNCaP cell proliferation and migration. Heparin affin regulatory peptide (HARP) or pleiotrophin seems to be an important mediator of FGF2 stimulatory effects, since the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, FGF2, through FGF receptors (FGFRs), significantly induced HARP expression and secretion by LNCaP cells and increased luciferase activity of a reporter gene vector carrying the full-length promoter of HARP gene. Using a combination of Western blot analyses, as well as genetic and pharmacological inhibitors, we found that activation of FGFR by FGF2 in LNCaP cells leads to NAD(P)H oxidase-dependent hydrogen peroxide production, phosphorylation of ERK1/2 and p38, activation of AP-1, increased expression and secretion of HARP, and, finally, increased cell proliferation and migration. These results establish the role and the mode of activity of FGF2 in LNCaP cells and support an interventional role of HARP in FGF2 effects, providing new insights on the interplay among growth factor pathways within prostate cancer cells.     

Sequential appearance of epidermal growth factor in plasma membrane-associated and intracellular vesicles during endocytosis

Receptor-mediated internalization of epidermal growth factor (EGF) occurs by a process involving initially clathrin-coated pits on the cell surface and the subsequent formation of ligand-containing endosomes. Using a modified acid wash technique, cell surface-bound EGF was removed. Utilizing sucrose density centrifugation, the residual cell-associated EGF was separated into plasma membrane-associated and intracellular vesicle-associated forms. Using these procedures we have identified a transient form of cell-associated EGF that is still attached to the plasma membrane but not accessible to the extracellular fluid. This form of EGF appears to be the precursor for endosomic EGF. We suggest that this intermediate form represents the receptor-ligand complex shown by electronmicroscopy to be located in narrow-necked plasma membrane invaginations (Willingham, M. C., and Pastan, I. (1980) Cell 21, 67-77).     

Heparin and heparan sulfate increase the radius of diffusion and action of basic fibroblast growth factor

The radius of diffusion of basic FGF (bFGF) in the presence and in the absence of the glycosaminoglycans heparin and heparan sulfate was measured. Iodinated 125I-bFGF diffuses further in agarose, fibrin, and on a monolayer of bovine aortic endothelial (BAE) cells in the presence of heparin than in its absence. Heparan sulfates affected the diffusion of 125I-bFGF in a manner similar to, though less pronounced than, heparin. When applied at the center of a monolayer of BAE cells, bFGF plus heparin stimulated morphological changes at a 10-fold greater radius than bFGF alone. These results suggest that bFGF-heparin and/or heparan sulfate complexes may be more effective than bFGF alone in stimulating cells located away from the bFGF source because the bFGF-glycosaminoglycan complex partitions into the soluble phase rather than binding to insoluble glycosaminoglycans in the extracellular matrix. Thus, the complex of bFGF and glycosaminoglycan may represent one of the active forms of bFGF in vivo.     

Granulocyte colony-stimulating factor potentiates anti-Candida albicans growth inhibitory activity of polymorphonuclear cells

Abstract Granulocyte colony-stimulating factor (G-CSF) stimulates a subset of granulocyte colony forming cells and when administered to neutropenic individuals results in recovery of blood neutrophil numbers to normal levels. Therefore, G-CSF may be a useful therapeutic agent for infections in immunocompromised hosts. However, to date there has been only limited information that G-CSF activates the antimicrobial activity of neutrophils. In the present study, we found that recombinant G-CSF promotes the anti- Candida albicans activity of normal human blood polymorphonuclear (PMN) cells in vitro using both a ³H-glucose uptake procedure and a Candida colony counting assay. As little as 0.1 ng/ml G-CSF induced significant anti-Candida activity in the PMN cultures. G-CSF treatment also enhanced superoxide anion production by the PMNs in response to f-MLP as determined by the superoxide dismutase inhibitable cytochrome C reduction method. Such results show that G-CSF can promote the antimicrobial activity of peripheral blood PMNs against C. albicans .     

Heparin modulates the growth and adherence and augments the growth-inhibitory action of TNF-alpha on cultured human keratinocytes

Previous works suggest the involvement of mast cells in the epithelialization of chronic wounds. Since heparin is a major mediator stored in the secretory granules of mast cells, the purpose of this work was to elucidate the function of heparin in epithelialization using in vitro culture models. For this, low- and high-calcium media in monolayer and epithelium cultures of keratinocytes were used. Also, an assay based on keratinocyte adherence onto plastic surface was used as well. Heparin (0.02-200 microg/ml) inhibited keratinocyte growth in a non-cytotoxic and dose-dependent manner in low- and high-calcium media, Keratinocyte-SFM and DMEM, in the absence of growth factors and serum. Also, heparin inhibited the growth of keratinocyte epithelium in the presence of 10% fetal calf serum and DMEM. Instead, in the presence of Keratinocyte-SFM and growth factors, heparin at 2 microg/ml inhibited the growth by 18% but at higher heparin concentrations the inhibition was reversed to baseline. TNF-alpha is another preformed mediator in mast cell granules and it inhibited keratinocyte growth in monolayer and epithelium cultures. Interestingly, heparin at 2-20 microg/ml augmented or even potentiated this growth-inhibitory effect of TNF-alpha. The association of TNF-alpha with heparin was shown by demonstrating that TNF-alpha bound tightly to heparin-Sepharose chromatographic material. However, heparin could not augment TNF-alpha-induced cell cycle arrest at G0/G1 phase or intercellular adhesion molecule-1 expression in keratinocytes. In the cell adherence assay, heparin at 2 microg/ml inhibited significantly by 12-13% or 33% the adherence of keratinocytes onto the plastic surface coated with fibronectin or collagen, respectively, but this inhibition was reversed back to baseline at 20 or 200 microg/ml heparin. Also, heparin affected the cell membrane rather than the protein coat on the plastic surface. In conclusion, heparin not only inhibits or modulates keratinocyte growth and adherence but it also binds and potentiates the growth-inhibitory function of TNF-alpha.     

Ethanol withdrawal tremor potentiates the tremorogenic action of nicotine

The tremorogenic effect of nicotine was studied in control rats and in rats withdrawn for 16-48 h from six to nine days' ethanol administration. The frequency and the intensity of tremor were measured electronically. A single dose of nicotine 5 mg/kg caused shortlasting (2-3 min) convulsions within 1 min after injection in control rats. Tremor occurred first after five repeated injections of nicotine (5 mg/kg) at half-hour intervals. This tremor encompassed peak frequencies of 6, 10 and 15 Hz. Hexamethonium at a dose of 10 mg/kg did not inhibit the tremor but eliminated the highest peak frequency (15 Hz) and tended to increase the intensity. Propranolol 5 mg/kg completely abolished the nicotine-induced tremor in control rats. Rats withdrawn from repeated ethanol administration showed a marked hypersensitivity to the tremorogenic action of nicotine so that a single dose of 1 mg/kg of nicotine caused clear tremor. The frequency spectra of this tremor showed peak frequencies at 6, 10 and 12 Hz. Hexamethonium did not change these frequencies. Furthermore, it tended to increase the intensity of nicotine-induced tremor in ethanol-withdrawn rats. Propranolol did not inhibit the tremor although it eliminated the 12 Hz peak frequency. The results suggest that the hypersensitivity to the tremorogenic action of nicotine in ethanol-withdrawn rats is not mediated by a sympathetic beta-adrenergic mechanism.     

Modulation of stimulatory action of follicle stimulating hormone (FSH) and inhibitory action of epidermal growth factor (EGF) on aromatase activity in Sertoli cells by calcium

Aromatization of testosterone by cultured Sertoli cells isolated from immature rats was stimulated more than 7-fold by follicle stimulating hormone (FSH) or dcAMP. The effects of FSH and dcAMP could be partly inhibited by epidermal growth factor (EGF) in a dose-dependent manner (ID500.5 nM). The phorbol ester 4 beta-phorbol-12-myristate-13-acetate (PMA) could also inhibit aromatase activity in a fashion similar to EGF. When 3 mM EGTA was present in the culture medium, the inhibitory effect of EGF was abolished but the stimulatory effect of FSH or dcAMP was magnified. These results suggest that EGF exerts a negative control on aromatase via calcium and protein kinase C. The abolishment of the inhibitory effect of EGF and the enhancement of the stimulatory effect of FSH or dcAMP by a calcium deficiency may be an indication that growth factors produced by Sertoli cells negatively controls FSH-induced responses in an autocrine fashion.     

Endurance exercise potentiates the stimulatory influence of oestrogen-progesterone on LH and FSH release in the rat

Ovariectomized rats were treated with oestradiol benzoate and progesterone or GnRH. Prolonged exercise (running 4 days per week for 6 weeks) markedly potentiated the oestrogen/progesterone-induced release of LH and FSH, but the pituitary response to an injection of GnRH was unaffected. In contrast, at 24 h after a single exercise bout there was no apparent effect on steroid and GnRH stimulated LH and FSH responses although an acute exercise session given on the day of the LH surge inhibited steroid-induced LH release in some rats. We conclude that strenuous, prolonged exercise-training in the ovariectomized rat seems to modify the ability of the hypothalamus to release GnRH. The results were not attributable to a single bout of exercise since the gonadotrophin responses immediately or 24 h after such exercise did not parallel the results observed in the trained rats.     

Self-association of the plasma membrane-associated clathrin assembly protein AP-2

A self-association reaction involving the plasma membrane-associated clathrin assembly protein AP-2 has been detected by incubating AP-2 alone under solution conditions that would favor the assembly of complete coat structures if clathrin were present. Self-association was rapid, unaffected by nonionic detergents, readily reversible, and gave rise to sedimentable aggregates. Only the AP subtype AP-2 exhibited self-association: the structurally or functionally related assembly proteins AP-1 and AP-3 and unrelated proteins neither self-associated nor were incorporated into the AP-2 aggregate. AP-2 interactions responsible for self-association were of high affinity, with an apparent Kd of approximately 10(-8)M. By proteolytic dissection, the self-association domain was localized to the core of the molecule containing the intact 50- and 16-kDa polypeptides in association with the truncated 60-66-kDa moieties of the parent alpha/beta polypeptides. Self-association of the intact AP-2 molecule was pH-dependent, exhibiting an apparent pKa approximately 7.4. While it is unlikely that the large AP-2 aggregates formed in solution are themselves biologically relevant structures, the AP-2 interactions involved in their formation have properties consistent with their occurrence in intact cells and thus may be important in cellular functions of the plasma membrane-localized assembly protein.