Biomedical subjects

D G Fernig

Publications and source records attributed to D G Fernig.

At least 37 records · Page 2Linked to original sources

PubMed2000

Stimulation of DNA synthesis and cell proliferation of human mammary myoepithelial-like cells by hepatocyte growth factor/scatter factor depends on heparan sulfate proteoglycans and sustained phosphorylation of mitogen-activated protein kinases p42/44.

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparan/dermatan sulfate-binding growth factor produced by stromal cells that acts as a paracrine effector on neighboring epithelia. HGF/SF stimulated DNA synthesis in human mammary (Huma) 109 myoepithelial-like cells grown on collagen I and fibronectin substrata but not when grown on plastic. Dual phosphorylation of mitogen-activated protein kinases (p42/44(MAPK)) was required for this stimulation of DNA synthesis. In Huma 109 cells cultured on plastic, HGF/SF stimulated a transient phosphorylation of p42/44(MAPK), which reached a maximum at 10 min after addition of the growth factor and returned to near basal levels after 20 min. In contrast, the phosphorylation of p42/44(MAPK) stimulated by HGF/SF in cells cultured on collagen I or fibronectin was sustained over 45 min. In Huma 109 cells deficient in sulfated glycosaminoglycans, HGF/SF failed to stimulate p42/44(MAPK) phosphorylation or DNA synthesis on any substratum, even when soluble heparan sulfate proteoglycans purified from the cells or from the culture medium were added. However, HGF/SF stimulated DNA synthesis and a sustained phosphorylation of p42/44(MAPK) in sulfated glycosaminoglycan-deficient Huma 109 cells plated on a substratum of medium HSPGs but not cell HSPGs. The HGF/SF-induced proliferation is thus highly dependent on heparan sulfate proteoglycans in myoepithelial-like cells.

Breast↗

PubMed2000

Differential regulation of FGF-1 and -2 mitogenic activity is related to their kinetics of binding to heparan sulfate in MDA-MB-231 human breast cancer cells.

The growth of the malignant human mammary MDA-MB-231 cells is stimulated by fibroblast growth factor-1 (FGF-1) but not by FGF-2. When these cells are cultured in the presence of chlorate, an inhibitor of heparan sulfate (HS) sulfation, their proliferation is stimulated by both FGF-1 and FGF-2. We analyzed the interactions of FGF-1 and FGF-2 with HS purified from the cell layer and the culture medium of control and chlorate-treated MDA-MB-231 cells. The HS from the cell layer bound FGF-1 with faster association kinetics than the HS from the culture medium, and so had a higher affinity for FGF-1. Chlorate treatment had no significant effect on the FGF-1 binding kinetics of the HS. In contrast to FGF-1, chlorate treatment of the cells significantly altered the FGF-2 binding kinetics. The HS from untreated cells possessed two binding sites for FGF-2, one with fast association kinetics (k(ass) 470,000 to 610,000 M(-1) s(-1)) and a high affinity (K(d) 46 to 70 nM) and one with slower association kinetics (k(ass) 74,000 to 100,000 M(-1) s(-1)) and a lower affinity (K(d) 290 to 400 nM). HS from chlorate-treated cells possessed just a single binding site for FGF-2 with fast association kinetics (k(ass) 270,000 to 290,000 M(-1) s(-1)) and a high affinity (K(d) 41 to 57 nM). These results show that there is a relationship between the binding kinetics of FGFs and their ability to stimulate cell growth.

Artifacts↗

PubMed2000

Presentation of IFN-gamma to nitric oxide-producing cells: a novel function for mast cells.

We report that mast cells can bind and present IFN-gamma in a functionally active form to macrophages. Flow-cytometric analysis revealed that biotinylated IFN-gamma bound equally well to purified peritoneal mast cells from both IFN-gammaR knockout and wild-type mice, indicating a non-IFN-gammaR binding site. Purified peritoneal mast cells, loaded with IFN-gamma for 30 min and washed, were able to induce NO synthesis by peritoneal macrophages. This response required cell contact and expression of IFN-gammaR on the responding macrophages, but not the mast cells. Human HMC-1 mast cells were also able to present IFN-gamma to mouse macrophages. Enzyme treatment of mouse mast cells revealed that binding of IFN-gamma was predominantly to chondroitin sulfate B (dermatan sulfate). Binding of IFN-gamma to dermatan sulfate was confirmed by inhibition ELISA. This study demonstrates for the first time that mast cells can present IFN-gamma to other cells via glycosaminoglycans. Mast cells may act as a reservoir of surface-stored functionally active cytokines.

Animals↗

PubMed2000

Heparan sulphate. Regulation of growth factors in the mammary gland.

Heparan sulphate (HS) is a linear glycosaminoglycan found ubiquitously on the surface and in the pericellular matrix of metazoan cells that is covalently attached to core proteins to form HS proteoglycans. HS interacts specifically with a large number (> 100) of extracellular regulatory proteins, many of which are key players in mammary development and function. HS thus acts a receptor, in which capacity it regulates the bioavailability, localisation and activity of these regulatory proteins.

Animals↗

PubMed2000

Heparin and in-vitro experimental lung hypoplasia.

Pulmonary hypoplasia (PH) is a leading contributor to the lethality of congenital diaphragmatic hernia (CDH). Studies now suggest that PH arises prior to visceral herniation. Growth factors (GF) are pivotal to this embryonic lung growth. With striking in-vitro effects on lung morphogenesis, GF are under investigation as therapies for PH. Heparin modulates the kinetics of heparan-sulphate binding ligands that drive lung development. We hypothesised that heparin may rescue PH by favourable alteration of endogenous pulmonary GF activity. Normal and hypoplastic lung primordia were microdissected on day 13.5 of gestation and cultured for up to 78 h in plain media with and without heparin. In-vitro morphological development was studied by serial measurements of terminal bud count, lung area, and lung perimeter. Nitrofen-exposed lungs cultured with heparin showed no significant improvements in terminal bud count, lung area, and lung perimeter at 30, 54, and 78 h compared to untreated hypoplastic lungs maintained in vitro. In normal lungs heparin demonstrated no sustained significant morphological effects compared to untreated control lungs. In this study, heparin did not stimulate branching morphogenesis of normal or hypoplastic lungs in our organ culture system. Known at higher concentrations to inhibit smooth-muscle proliferation, heparin may ameliorate pulmonary vascular hypermuscularisation with the prospect of benefiting CDH infants on extracorporeal membrane oxygenation. Future studies will address the impact of exogenous GF on hypoplastic lung development in organ culture.

Animals↗

PubMed2000

Early lung malformations in congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Lung hypoplasia, a leading contributor to the lethality of congenital diaphragmatic hernia (CDH), has been attributed to compression of the fetal lung by herniated abdominal viscera. Contested findings in experimental CDH suggest that lung malformation may precede diaphragmatic hernia. To address this unresolved question, we studied the pattern and progress of embryonic lung development in vivo and in vitro before diaphragmatic herniation in the nitrofen CDH model. METHODS: Sprague-Dawley rats were fed nitrofen on day 9.5 of pregnancy to induce pulmonary hypoplasia and CDH in newborns (term, day 22). Control rats received olive oil. Embryonic lungs were microdissected on day 13.5 gestation, 24 hours after lung primordia bud from the foregut (normal diaphragmatic closure, day 16.5). In vivo airway branching was measured by counting terminal lung buds at this stage. Lungs were cultured for up to 78 hours and longitudinal in vitro development studied by serial measurements of terminal bud count, area, and perimeter. RESULTS: At 13.5 days of gestation in vivo, nearly 99% of normal lungs (n = 130) had > or = 6 terminal lung buds. In contrast, 36% of the nitrofen-exposed lungs (n = 170) fell short of this developmental milestone with less than 6 terminal buds (P < .001). In vitro, the nitrofen lungs had reduced area compared with controls after 6, 30, and 54 hours (P = .001, P < .001, and P = .001, respectively). Bud count and epithelial perimeter were reduced in the nitrofen lungs after 6 and 30 hours in vitro (P < .001 and P = .01 v normal terminal bud count; P < .001 and P= .002 v normal perimeter). CONCLUSIONS: Before diaphragmatic herniation, nitrofen-exposed embryos in vivo have a 36% frequency of reduced airway branching that strikingly parallels the 30% to 40% term incidence of CDH in this model. The authors propose that this early lung anomaly is restricted to a subset of nitrofen-exposed embryos in which it is integral to an emerging CDH phenotype. In vitro data confirm that nitrofen-induced pulmonary hypoplasia precedes visceral herniation and persists to allow experimental manipulation in culture. The developmental biology underlying lung hypoplasia in CDH may now be addressed using this organ culture system.

Animals↗

PubMed2000

Cell proliferation and apoptosis in experimental lung hypoplasia.

BACKGROUND/PURPOSE: Current treatment for lethal pulmonary hypoplasia in congenital diaphragmatic hernia (CDH) may be hampered by uncertainty over its origin. Herniation of abdominal organs into the chest was thought to produce lung hypoplasia by compression. Experimental CDH models suggest that disturbed lung growth precedes these events. Mammalian development comprises cell differentiation, proliferation and programmed cell death or apoptosis. Could lung hypoplasia in CDH result from alterations in these processes well before visceral herniation takes place? The aim of this study was to compare cell proliferation and apoptosis in normal and hypoplastic embryonic lungs before normal diaphragmatic closure using a CDH model. METHODS: Sprague-Dawley rats were given 100 mg of nitrofen on day 9.5 of pregnancy to create lung hypoplasia and CDH in newborns (term, 22 days). Control rats received olive oil. Cell proliferation in embryonic lung specimens was measured by bromodeoxyuridine (BrdU) incorporation at 13.5 to 15.5 days' gestation, before normal diaphragmatic closure in this species (day 16.5). Apoptosis was measured by the in situ end-nick labelling (TUNEL) method in lung sections obtained from rat embryos of 13.5 to 16.5 days' gestation. RESULTS: High levels of cell proliferation were seen in both normal control and nitrofen-exposed lungs. However, 24 hours before normal diaphragmatic closure, nitrofen-exposed lungs had significant reductions in cell proliferation on day 15.5 of gestation (P = .009 v controls). Apoptosis occurred at low levels throughout the developmental stages examined (< 0.3%) without significant differences encountered between the study groups. CONCLUSIONS: These findings have shown high rates of cell division during normal lung development before diaphragmatic closure. Decreased levels over this critical period in gestation may contribute to early lung anomalies in the nitrofen CDH model. Strategies to promote cell proliferation in the fetal lung may therefore hold future promise in human CDH. Apoptosis appears not to play a major role in hypoplastic lung development. Therapies to inhibit apoptosis would seem unlikely to improve this early lung growth.

Animals↗

PubMed2000

Intracellular trafficking and release of intact edible mushroom lectin from HT29 human colon cancer cells.

Our previous studies have shown that the Galbeta1-3GalNAcalpha- (Thomsen-Friedenreich antigen)-binding lectin from the common edible mushroom Agaricus bisporus (ABL) reversibly inhibits cell proliferation, and this effect is a consequence of inhibition of nuclear localization sequence-dependent nuclear protein import after ABL internalization [Yu, L.G., Fernig, D.G., White, M.R.H., Spiller, D.G., Appleton, P., Evans, R.C., Grierson, I., Smith, J.A., Davies, H., Gerasimenko, O.V., Petersen, O.H., Milton, J.D. & Rhodes, J.M. (1999) J. Biol. Chem. 274, 4890-4899]. Here, we have investigated further the intracellular trafficking and fate of ABL after internalization in HT29 human colon cancer cells. Internalization of 125I-ABL occurred within 30 min of the lectin being bound to the cell surface. Subcellular fractionation after pulse labelling of the cells with 125I-ABL for 2 h at 4 degrees C followed by culture of the cells at 37 degrees C demonstrated a steady increase in radioactivity in a crude nuclear extract. The radioactivity in this extract reached a maximum after 10 h and declined after 20 h. Release of ABL from the cell, after pulse labelling, was assessed using both fluorescein isothiocyanate-labelled ABL and 125I-ABL and was slow, with a t1/2 of 48 h. Most of the 125I-ABL both inside cells and in the medium remained intact, as determined by trichloroacetic acid precipitation and SDS/PAGE, and after 48 h only 22 +/- 2% of ABL in the medium and 14 +/- 2% inside the cells was degraded. This study suggests that the reversibility of the antiproliferative effect of ABL is associated with its release from cells after internalization. The internalization and subsequent slow release, with little degradation of ABL, reflects the tendency of lectins to resist biodegradation and implies that other endogenous or exogenous lectins may be processed in this way by intestinal epithelial cells.

Agaricales↗

PubMed2000

In vitro effects of growth factors on lung hypoplasia in a model of congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Pulmonary hypoplasia, a leading contributor to the lethality of congenital diaphragmatic hernia (CDH), precedes diaphragmatic malformation in the nitrofen model and persists to allow experimental manipulations in organ culture. Fibroblast growth factors (FGFs) are crucial to early lung development. Acidic FGF (FGF-1) binds to all FGF receptors and enhances in vitro branching morphogenesis. Basic FGF (FGF-2) is localized to developing airway epithelium, basement membrane, and extracellular matrix. Heparin (HEP) modulates FGF kinetics and inhibits smooth muscle proliferation in lung primordia. The aim of this study was to examine the morphological effects of fibroblast growth factors and heparin on lung hypoplasia in an organ culture model. METHODS: Sprague-Dawley rats were fed nitrofen on day 9.5 of pregnancy to induce lung hypoplasia and CDH in newborns. Control rats received olive oil. Normal and hypoplastic lung primordia were microdissected on day 13.5 of gestation and cultured up to 78 hours in plain media with or without FGF-1 or FGF-2, with or without HEP. In vitro morphological development was studied by serial measurements of terminal bud count, lung area, and lung perimeter. RESULTS: Over 120 fetal lung specimens were studied (n > or = 4 per group). Significant increases in area, perimeter, and bud count were seen in normal lungs cultured with FGF-1 plus HEP compared with control media (P < .05). In the nitrofen lungs, FGF1 plus HEP yielded reductions in all parameters compared with those in control media (P < .05), whereas FGF-2 produced significant expansion in lung area but marked reductions in bud count and lung perimeter divided by square root of area (P < .05). Heparin did not produce substantial or sustained alteration of morphology in normal or hypoplastic lungs. CONCLUSIONS: These observations may indicate an intrinsic abnormality of FGF processing in the hypoplastic nitrofen lung before diaphragmatic malformation. Heparin did not rescue abnormal lung development. Mechanisms underlying the differential effects of these agents now need to be explored to target fetal lung growth and improve the dismal prognosis of human CDH.

Animals↗

PubMed2000

Human lactoferrin interacts with soluble CD14 and inhibits expression of endothelial adhesion molecules, E-selectin and ICAM-1, induced by the CD14-lipopolysaccharide complex.

Lipopolysaccharides (LPS), either in the free form or complexed to CD14, a LPS receptor, are elicitors of the immune system. Lactoferrin (Lf), a LPS-chelating glycoprotein, protects animals against septic shock. Since optimal protection requires administration of Lf prior to lethal doses of LPS, we hypothesized that interactions between Lf and soluble CD14 (sCD14) exist. In a first step, human sCD14 and human Lf (hLf) were used to determine the kinetic binding parameters of hLf to free sCD14 in an optical biosensor. The results demonstrated that hLf bound specifically and with a high affinity (K(d) = 16+/-7 nM) to sCD14. Affinity chromatography studies showed that hLf interacted not only with free sCD14 but also, though with different binding properties, with sCD14 complexed to LPS or lipid A-2-keto-3-deoxyoctonic acid-heptose. In a second step, we have investigated whether the capacity of hLf to interact with sCD14 could modulate the expression of endothelial-leukocyte adhesion molecule 1 (E-selectin) or intercellular adhesion molecule 1 (ICAM-1) induced by the sCD14-LPS complex on human umbilical vein endothelial cells (HUVEC). Our experiments show that hLf significantly inhibited both E-selectin and ICAM-1 expressions at the surface of HUVEC. In conclusion, these observations suggest that the anti-inflammatory effects of hLf are due not only to the ability of the molecule to chelate LPS but also to its ability to interact with sCD14 and with the sCD14 complexed to LPS, thus modifying the activation of endothelial cells.

Amino Acid Sequence↗

PubMed1999

Modulation of mammary development and programmed cell death by the frequency of milk removal in lactating goats.

1. Unilateral changes in mammary cell number are elicited when one gland is milked more or less frequently than the contralateral gland in lactating goats. These changes were investigated using histochemical and immunocytochemical markers of mammary cell types, and the degree of mammary apoptosis was determined by end-labelling of fragmented DNA. 2. Histological analysis confirmed that unilateral cessation of milking initiated involution and cell loss preferentially in the unmilked gland. The presence of fragmented DNA and morphological characteristics consistent with apoptosis demonstrated that these changes in mammary cell number in unmilked glands were, in part, the result of programmed alveolar cell death. 3. De-differentiation of the remaining secretory cells to ductal epithelial cells occurred with an increase in staining of cytokeratin markers and decreased staining by peanut lectin and casein antisera. 4. Differential once- and thrice-daily milking of lactating goats was also associated with unilateral changes in mammary cell number and milk yield. Milk yield and alveolar size were reduced after 4 weeks of infrequent milking. The latter was due to the increased loss of secretory cells by apoptosis, as indicated by a higher degree of fragmented DNA laddering. 5. After 10 weeks of differential milking, a homogeneous secretory morphology, albeit with smaller alveoli, was maintained in thrice-daily milked glands. Once-daily milked glands possessed a heterogeneous composition of terminal structures, resulting in the simultaneous presence of secretory and involuting alveoli as well as resting ductules. 6. The differences in programmed cell death and mammary morphology between unmilked and twice-daily milked glands, and between once- and thrice-daily milked glands, suggests that mammary apoptosis is subject to modulation by intra-mammary mechanisms sensitive to the frequency of milk removal.

Animals↗

PubMed1999

Glycosaminoglycans differentially bind HARP and modulate its biological activity.

Heparin affin regulatory peptide (HARP) is a polypeptide belonging to a family of heparin binding growth/differentiation factors. The high affinity of HARP for heparin suggests that this secreted polypeptide should also bind to heparan sulfate proteoglycans derived from cell surface and extracellular matrix defined as extracellular compartments. Using Western blot analysis, we detected HARP bound to heparan sulfate proteoglycans in the extracellular compartments of MDA-MB 231 and MC 3T3-E1 as well as NIH3T3 cells overexpressing HARP protein. Heparitinase treatment of BEL cells inhibited HARP-induced cell proliferation, and the biological activity of HARP in this system was restored by the addition of heparin. We report that heparan sulfate, dermatan sulfate, and to a lesser extent, chondroitin sulfate A, displaced HARP bound to the extracellular compartment. Binding analyses with a biosensor showed that HARP bound heparin with fast association and dissociation kinetics (kass = 1.6 x 10(6) M-1 s-1; kdiss = 0.02 s-1), yielding a Kd value of 13 nM; the interaction between HARP and dermatan sulfate was characterized by slower association kinetics (kass = 0.68 x 10(6) M-1 s-1) and a lower affinity (Kd = 51 nM). Exogenous heparin, heparan sulfate, and dermatan sulfate potentiated the growth-stimulatory activity of HARP, suggesting that corresponding proteoglycans could be involved in the regulation of the mitogenic activity of HARP.

3T3 Cells↗

PubMed1999

Edible mushroom (Agaricus bisporus) lectin, which reversibly inhibits epithelial cell proliferation, blocks nuclear localization sequence-dependent nuclear protein import.

The Galbeta1-3GalNAcalpha (TF antigen)-binding lectin (ABL) from the common edible mushroom (Agaricus bisporus) has a potent anti-proliferative effect without any apparent cytotoxicity. This unusual combination of properties prompted investigation of its mechanism of action. In contrast to soluble lectin, agarose-immobilized, and hence noninternalizable ABL had no effect on proliferation of HT29 colon cancer cells. Electron microscopy of HT29 cells incubated with fluorescein- and gold-conjugated ABL showed internalization of the lectin into endocytotic vesicles and multivesicular bodies. Confocal microscopy showed perinuclear accumulation of fluorescein isothiocyanate-conjugated lectin, which also inhibits HT29 cell proliferation, raising the possibility that the lectin might interfere with nuclear pore function. Transport of heat shock protein 70 into the nucleus in response to heat shock was blocked by preincubation of HT29 cells for 6 h with 40 micrograms/ml ABL. In digitonin-permeabilized cells, nuclear uptake of bovine albumin conjugated to a nuclear localization sequence (NLS)-containing peptide was also inhibited by a 15-min preincubation with 40-100 micrograms/ml ABL. In contrast, serum-stimulated nuclear translocation of mitogen-activated protein kinase, which is NLS-independent, was not affected by pretreatment of cells with the lectin. These results suggest that the anti-proliferative effect of ABL is likely to be a consequence of the lectin trafficking to the nuclear periphery, where it blocks NLS-dependent protein uptake into the nucleus.

Adult↗

PubMed1999

Identification of cell types in the developing goat mammary gland.

The goat was chosen as the model system for investigating mammary gland development in the ruminant. Histological and immunocytochemical staining of goat mammary tissue at key stages of development was performed to characterize the histogenesis of the ruminant mammary gland. The mammary gland of the virgin adult goat consisted of a ductal system terminating in lobules of ductules. Lobuloalveolar development of ductules occurred during pregnancy and lactation which was followed by the regression of secretory alveoli at involution. The ductal system was separated from the surrounding stroma by a basement membrane which was defined by antisera raised against laminin and Type IV collagen. Vimentin, smooth-muscle actin and myosin monoclonal antisera as well as antisera to cytokeratin 18 and multiple cytokeratins stained a layer of myoepithelial cells which surround the ductal epithelium. Staining of luminal epithelial cells by monoclonal antibodies to cytokeratins was dependent on their location along the ductal system, from intense staining in ducts to variable staining in ductules. The staining of epithelial cells by monoclonals to cytokeratins also varied according to the developmental status of the goat, being maximal in virgin and involuting glands, lowest at lactation and intermediate during gestation. In addition, cuboidal cells, situated perpendicular to myoepithelial cells and adjacent to alveolar cells in secretory alveoli, were also stained by cytokeratin monoclonal antibodies and antisera to the receptor protein, erbB-2, in similar fashion to luminal epithelial cells. These results demonstrate that caprine mammary epithelial cell differentiation along the alveolar pathway is associated with the loss of certain types of cytokeratins and that undifferentiated and secretory alveolar epithelial cells are present within lactating goat mammary alveoli.

Animals↗

PubMed1998

Hepatocyte growth factor/scatter factor has distinct classes of binding site in heparan sulfate from mammary cells.

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparan sulfate (HS)-binding growth factor and morphogen for mammary epithelial cells that is produced by mammary stromal fibroblasts. HS chains, purified as peptidoglycans from a panel of cell lines representative of the ductal epithelial cell (Huma 123), the myoepithelial cell (Huma 109), the stromal fibroblast (Rama 27), and malignant mammary epithelial cells (MCF-7 and ZR-75), were used in a biosensor-based assay to identify the classes of HGF/SF-binding sites in the polysaccharide chains. At least three distinct binding sites were identified. One site exhibits fast association and fast dissociation kinetics [kass (1.4-7.7) x 10(6) M-1 s-1; kdiss 0. 0032-0.0096 s-1] and is present on the HS from benign Huma 123 epithelial cells, Huma 109 myoepithelial-like cells, and ZR-75 malignant cells. The second binding site, found on HS from the malignant MCF-7 cells, has slower HGF/SF-binding kinetics (kass 0.20 x 10(6) M-1 s-1; kdiss 0.00055 s-1). The third binding site possesses fast association and slow dissociation kinetics (kass 1.1 x 10(6) M-1 s-1; kdiss 0.00020 s-1) and was found on the HS isolated from the culture medium of the Huma 123 benign epithelial cells. The first and second binding sites have a similar Kd, 1-3 nM, while the third binding site has a considerably higher affinity for HGF/SF (Kd 200 pM). The three binding sites seem to be mutually exclusive, since each sample of HS possessed just one of the sites.

Animals↗

PubMed1998

Interaction of heparan sulfate from mammary cells with acidic fibroblast growth factor (FGF) and basic FGF. Regulation of the activity of basic FGF by high and low affinity binding sites in heparan sulfate.

We have determined the relationship between the binding sites for acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) in heparan sulfate (HS) prepared from a panel of mammary cell lines and the ability of the HS to activate aFGF and bFGF in DNA synthesis assays. The ka of the HS for aFGF fell into three groups, whereas the kd (0.0015-0.016 s-1) and the Kd (0.4-8.6 microM) formed a continuum. bFGF possessed a high affinity binding site (Kd 22-30 nM) with a fast ka (320,000-550,000 M-1 s-1), termed "fast/high," and a lower affinity site (Kd 47-320 nM) with a slower ka (35,000-150,000 M-1 s-1), termed "slow/low." Most of the species of HS possessed the latter binding site, which was able to activate bFGF in HS-deficient fibroblasts. However, the HS from the culture medium of the mammary fibroblasts and the myoepithelial-like cells possessed both a fast/high and a slow/low binding site and could not activate bFGF, although it could potentiate the growth-stimulatory activity of aFGF. Treatment of the HS possessing two binding sites for bFGF with heparitinase 1 released oligosaccharides that were able to restore the activity of bFGF in HS-deficient fibroblasts.

Animals↗

PubMed1998

Hepatocyte growth factor/scatter factor binds with high affinity to dermatan sulfate.

We have demonstrated by affinity chromatography that hepatocyte growth factor/scatter factor (HGF/SF) binds strongly to dermatan sulfate (DS), with a similar ionic strength dependence to that previously seen with heparan sulfate (HS). Analysis of binding kinetics on a biosensor yields an equilibrium dissociation constant, KD, of 19.7 nM. This corresponds to a 10-100-fold weaker interaction than that with HS, primarily due to a faster dissociation rate of the complex. The smallest DS oligosaccharide with significant affinity for HGF/SF by affinity chromatography appears to be an octasaccharide. A sequence comprising unsulfated iduronate residues in combination with 4-O-sulfated N-acetylgalactosamine is sufficient for high affinity binding. The presence of 2-O-sulfation on the iduronate residues does not appear to be inhibitory. These observations concur with our previous suggestions, from analyses of HS binding (Lyon, M., Deakin, J. A., Mizuno, K., Nakamura, T., and Gallagher, J.T. (1994) J. Biol. Chem. 269, 11216-11223), that N-sulfation of hexosamines and 2-O-sulfation of iduronates are not absolute requirements for glycosaminoglycan binding to HGF/SF. This is the first described example of a high affinity interaction between a growth factor and DS, and is likely to have significant implications for the biological activity of this paracrine-acting factor.

3T3 Cells↗

PubMed1998

Interactions of putative heparin-binding domains of basic fibroblast growth factor and its receptor, FGFR-1, with heparin using synthetic peptides.

We have examined structure-function relationships that have been proposed to account for the heparin-binding properties of basic fibroblast growth factor and its receptor, FGFR-1, using synthetic peptides, DNA synthesis assays and binding assays in a resonant mirror biosensor. The results suggest that the interaction of FGFR-1 with heparin may not be physiologically relevant and that the site of interaction of the polysaccharide on bFGF is more complex than has been anticipated.

Animals↗