Nerve dependent sulphated glycosaminoglycan synthesis in limb regeneration of the newt Pleurodeles waltl

Denervation of the amputated limb of newts stops the regeneration process by decreasing blastema cell proliferation. We investigated the effect of the denervation on each of the two compartments (epidermal cap, mesenchyme) in mid-bud blastemas on the level of sulphated glycosaminoglycans (GAGS). Denervation resulted in an increase of about threefold in the incorporation of [³⁵S] sulphate into mesenchyme GAGs but had no effect on the epidermal cap. The increase of GAG synthesis in the mesenchymal part of the blastema involved both heparan sulphates and chondroitin-dermatan sulphates. Gel filtration showed no change in GAGs size after denervation. These results confirm that the mesenchymal part of the mid-bud blastema is the main target of nerves and, as heparan sulphates are known to store acidic fibroblast growth factor (aFGF), a polypeptide found in the blastema (Boilly et al.. 1991), this suggest that the nerves' effect on glycosaminoglycans turnover could be implicated in the control of bioavailability of this growth factor in the blastema.     

In vitro control of blastema cell proliferation by extracts from epidermal cap and mesenchyme of regenerating limbs of axolotls

Summary The presence of a mitogenic activity in limb blastemas of axolotls was detected in crude extracts of blastemas at the mid-bud stage. The mitogenicity of the extracts was estimated from the mitotic index of blastema cells grown for 6 days in the presence of limb blastema extracts, with colchicine present for the last 2 days. All the extracts tested (whole blastema, blastemal mesenchyme, epidermal cap) significantly enhanced proliferation of blastema cells. The highest stimulation factors we observed were 7 × with 7 g protein/ml whole blastema extracts, 5.2 × with 14 g/ml blastemal mesenchyme extracts, and 11 x with 3.5 g/ml epidermal cap extracts. Hence the epidermal cap extracts appeared to be the most mitogenic. Extracts from the blastemal mesenchyme, although less mitogenic than the epidermal cap extracts, were more potent than nerve extracts [Albert P, Boilly B (1986) Biol Cell 58:251–262]. These results are discussed with regard to the production of growth factors during limb regeneration.     

Modulation of growth of human carcinoma SW-13 cells by heparin and growth factors

This study reports on the effects of heparin, basic and acidic fibroblast growth factors (bFGF and aFGF, respectively), and transforming growth factor type-e (TGFe) on the growth of a human adrenocortical carcinoma cell line, SW-13. Heparin has previously been shown to inhibit growth in several cell types, including smooth muscle cells, certain fibroblasts, and epithelial cells, and to modulate the effects of fibroblast growth factors. Whereas bFGF and aFGF bind tightly to heparin and elute from a heparin-Sepharose column with 2 M NaCl and 1.6 M NaCl, respectively, TGFe binds to heparin with lower affinity and can be eluted from heparin-Sepharose column with 0.5 M NaCl. TGFe is a polypeptide unrelated to FGF, is present in neoplastic and nonneoplastic tissues, and stimulates the growth of certain epithelial cells and fibroblasts in soft agar and monolayer. Since the growth of SW-13 cells is stimulated by TGFe and by bFGF, we hypothesized that heparin would inhibit the growth of SW-13 cells by binding to these growth factors and that the effects of heparin could be overcome with the addition of either growth factor. Our experiments confirmed that heparin inhibits the growth of SW-13 cells. A dose-dependent growth inhibition was observed in both monolayer and soft agar. The inhibition in monolayer was partially reversed upon heparin withdrawal. The effects of heparin in both monolayer and soft agar were at least partially overcome by TGFe and by basic or acidic FGF. Overall protein synthesis does not appear to be affected by heparin as measured by [35S]methionine uptake. In contrast, epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) were unable to overcome heparin-induced inhibition both in monolayer and in soft agar. Heparin also inhibited [3H]thymidine incorporation in AKR-2B and partially inhibited AKR-2B cell stimulation by TGFe; however, it further potentiated the already potent stimulation by bFGF. We propose that heparin, TGFe, bFGF, and aFGF modulate the growth of SW-13 cells and possibly of other epithelial cells in complex ways and that heparin-like substances present in the extracellular matrix play an important role in the control of epithelial growth.     

Endothelial cell growth factors in embryonic and adult chick brain are related to human acidic fibroblast growth factor.

We have investigated the nature of endothelial cell growth factors in 14-day embryonic and adult chick brain extracts. Mitogenic activity was isolated by a combination of cation-exchange, heparin-Sepharose affinity, and reverse-phase HPLC. Two major mitogenic fractions eluted from heparin-Sepharose at 0.8-1.3 M and 1.5-2 M. Biologically active proteins eluting at 0.8-1.3 M NaCl, after purification to homogeneity from embryonic and adult brain, were found to possess the same amino-terminal sequence as human acidic fibroblast growth factor (aFGF). The notion that the isolated mitogens represent chick aFGF is further supported by the findings that their affinity for heparin and their retention behavior in highly resolutive HPLC are indistinguishable from those of genuine aFGF. Mitogenic activities eluting at 1.5-2 M NaCl were also present in embryonic and adult brain, but in quantities insufficient for preliminary characterization. The high specific mitogenic activity for endothelial cells, high affinity for heparin and cross-reactivity with antibodies against bovine basic FGF (bFGF) suggest a relationship of those materials with basic FGF. Our data also suggest that the sequence of aFGF is highly conserved among vertebrates. While angiogenesis occurs predominantly in the embryonic brain, the absence of notable differences in the contents of the potent angiogenic factors aFGF and bFGF in embryonic versus adult chick brain is interesting.     

Heparin-derived oligosaccharides: affinity for acidic fibroblast growth factor and effect on its growth-promoting activity for human endothelial cells

The minimal structural requirements for the interaction of heparin with acidic fibroblast growth factor (aFGF) were investigated. Oligosaccharides (tetra- to decasaccharides) obtained by nitrous acid depolymerisation of standard heparin were separated by affinity chromatography on Sepharose-immobilised aFGF. The shortest fragment retained by the affinity column at 0.2 M NaCl and eluted at 1 M NaCl was a "regular" hexasaccharide, a trimer of the most abundant disaccharide sequence in heparin. More complex octa- and decasaccharides were also retained by the column. The oligosaccharides eluted by 1 M NaCl from the affinity column ("high-affinity" oligosaccharides) and those washed from the column at 0.2 M NaCl ("low-affinity" oligosaccharides) were compared for their capacity to protect aFGF from proteolysis and to potentiate its mitogenic activity. At a low ionic strength, all oligosaccharides tested, except the "regular" disaccharide, protected aFGF against trypsin and collagenase digestion. At higher ionic strength (greater than 0.2 M NaCl), only high-affinity oligosaccharides showed a protective effect. The high-affinity oligosaccharides (hexa- to decasaccharides) potentiated the mitogenic activity of aFGF, as measured by [3H]thymidine incorporation into DNA of human fibroblasts. The effect of the oligosaccharides on human endothelial cell proliferation was more complex: inhibition of proliferation was observed in the presence of serum and low concentrations of aFGF (1-5 ng/ml) and potentiation in the presence of higher concentrations of aFGF. The potentiating effect increased as a function of molecular size of the heparin fragments and, for a given size, as a function of the anionic charge of the oligosaccharide. Our results suggest that inhibition of cell proliferation by heparin may result from interference with an autocrine basic FGF-like activity.     

Effect of heparin on the binding affinity of acidic FGF for the cloned human FGF receptors, flg and bek.

Heparin potentiates the mitogenic activity of acidic fibroblast growth factor (aFGF) by 20-100 fold but mechanisms detailing this potentiation have not yet been fully elucidated. We report that heparin increases the binding affinity of aFGF for the two cloned and overexpressed human FGF receptors, flg and bek, by 2-3 fold. This increase in binding affinity, together with previous data demonstrating a 3-5 fold increase in the stability of aFGF, are likely to account for a significant portion of heparin's potentiation of aFGF activity observed in biological assay systems.     

Expression and biological effect of urodele fibroblast growth factor 1: relationship to limb regeneration

Fibroblast growth factors (FGFs) have been previously implicated in urodele limb regeneration. Here, we examined expression of FGF-1 by blastema cells and neurons and investigated its involvement in wound epithelial formation and function and in the trophic effect of nerves. Neurons innervating the limb and blastema cells in vivo and in vitro expressed the FGF-1 gene. The peptide was present in blastemas in vivo. Wound epithelium thickened when recombinant newt FGF-1 was provided on heparin-coated beads, demonstrating that the FGF-1 was biologically active and that the wound epithelium is a possible target tissue of FGF. FGF-1 did not stimulate accessory limb formation. FGF-1 was as effective as 10% fetal bovine serum in maintaining proliferative activity of blastema cells in vitro but was unable to maintain growth of denervated, nerve-dependent stage blastemas when provided on beads or by injection. FGF-1 had a strong stimulating effect on blastema cell accumulation and proliferation of limbs inserted into the body cavity that were devoid of an apical epithelial cap (AEC). These results show that FGF-1 can signal wound epithelium cap formation and/or function and can stimulate mesenchyme accumulation/proliferation in the absence of the AEC but that FGF-1 is not directly involved in the neural effect on blastema growth.     

Characterization of the receptor for keratinocyte growth factor. Evidence for multiple fibroblast growth factor receptors

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. KGF exhibits potent mitogenic activity for a variety of epithelial cell types but is distinct from other known FGFs in that it is not mitogenic for fibroblasts or endothelial cells. We report saturable specific binding of 125I-KGF to surface receptors on intact Balb/MK mouse epidermal keratinocytes. 125I-KGF binding was completed efficiently by acidic FGF (aFGF) but with 20-fold lower efficiency by basic FGF (bFGF). The pattern of 125I-acidic FGF binding and competition on Balb/MK keratinocytes and NIH/3T3 fibroblasts suggests that these cell types possess related but distinct FGF receptors. Scatchard analysis of 125I-KGF binding suggested major and minor high affinity receptor components (KD = 400 and 25 pM, respectively) as well as a third high capacity/low affinity heparin-like component. Covalent affinity cross-linking of 125I-KGF to its receptor on Balb/MK cells revealed two species of 115 and 140 kDa. KGF also stimulated the rapid tyrosine phosphorylation of a 90-kDa protein in Balb/MK cells but not in NIH/3T3 fibroblasts. Together these results indicate that Balb/MK keratinocytes possess high affinity KGF receptors to which the FGFs may also bind. However, these receptors are distinct from the receptor(s) for aFGF and bFGF on NIH/3T3 fibroblasts, which fail to interact with KGF.     

Nerve–blastema interactions induce fibroblast growth factor-1 release during limb regeneration in Pleurodeles waltl

Previous studies have shown that both fibroblast growth factor (FGF)-1 and nerves play an important function during limb regeneration, but no correlation between these two regeneration factors has yet been demonstrated. In the present study we first establish that exogenous FGF-2, a member of the FGF family that binds to the same high-affinity receptors as FGF-1, is able to stimulate both [³H]-thymidine incorporation and the mitotic index in the mesenchyme and the epidermal cells of denervated blastemas. We then use cocultures of spinal cord and blastema on heparin-coated dishes, an in vitro system mimicking the in vivo interactions during limb regeneration, to show that interactions between nerve fibers from the spinal cord and the blastema enhance the release of bioactive FGF-1. Release of this growth factor seemed to correlate with nerve fiber regeneration, as it decreased in the presence of the dipeptide Leu-Ala, known to inhibit neurite outgrowth, while the inverse dipeptide Ala-Leu was inactive. Therefore, these results support our hypothesis that the interaction between nervous tissue and blastema is permissive for the release of FGF-1, which in turn stimulates blastema cell proliferation.     

Protamine sulfate inhibits mitogenic activities of the extracellular matrix and fibroblast growth factor, but potentiates that of epidermal growth factor

Protamine sulfate, an inhibitor of angiogenesis in vivo, markedly inhibits the ability of angiogenic factors such as acidic or basic fibroblast growth factor (aFGF, bFGF) to stimulate the proliferation in vitro of either BHK-21 cells or vascular endothelial cells. The inhibition is reversible, and cells remain viable even after prolonged exposure to protamine sulfate. Protamine sulfate inhibits the mitogenic effects of both growth factors by preventing them from binding to their common cell surface receptors. It also inhibits the mitogenic activity of the extracellular matrix produced by bovine corneal endothelial cells. This substrate has been shown in previous studies to replace the requirement for FGF of many cell types. In contrast, protamine sulfate potentiates the mitogenic activity of epidermal growth factor (EGF). This indicates that protamine sulfate also acts at cellular sites which are not associated with FGF receptors.     

Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line.

We have characterized two high affinity acidic fibroblast growth factor (aFGF) receptors in a rat parathyroid cell line (PT-r). Affinity labeling with 125I-aFGF showed that these two receptors, apparent molecular masses, 150 and 130 kDa, respectively, display higher affinity for aFGF than for bFGF. The 150-kDa receptor bears a heparan sulfate chain(s), demonstrated by a decrease in size of 15-20 kDa with heparitinase digestion after affinity labeling. Heparitinase digestion before affinity labeling markedly reduced the intensity of the 150 kDa species. Scatchard analysis showed two different high affinity binding sites (Kd of 3.9 pM with 180 sites/cell and Kd of 110 pM with 5800 sites/cell). The higher affinity site was completely eliminated by digestion with heparitinase before adding labeled aFGF; the lower affinity site was unaffected. In ion exchange chromatography after metabolic labeling of the cells with [3H]glucosamine and affinity labeling with 125I-aFGF, the larger receptor-ligand complex, 165 kDa, eluted with approximately 0.5 M NaCl, typical eluting conditions for heparan sulfate proteoglycans. Both of the receptor-ligand complexes were smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than two major heparan sulfate proteoglycans, HSPG I and II, which we characterized in this cell line previously (Yanagishita, M., Brandi, M. L., and Sakaguchi, K. (1989) J. Biol. Chem. 264, 15714-15720). Both receptors have similar N-linked oligosaccharide and sialic acid contents, shown by analysis of affinity-labeled receptors upon digestion with glycopeptidase F and with neuraminidase. All together, these results suggest that PT-r cells bear two distinct high affinity receptors for aFGF, a 150-kDa receptor which is a heparan sulfate proteoglycan and another that is a glycoprotein. The heparan sulfate glycosaminoglycan moiety of the 150- kDa receptor is critical for high affinity binding of aFGF. These findings contrast with current concepts derived from other systems, suggesting that heparan sulfate glycosaminoglycans/proteoglycans function as a reservoir source for FGF or as a group of low affinity binding sites.     

Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor

Keratinocytes and fibroblasts isolated from human neonatal foreskin can be plated and grown through multiple rounds of division in vitro under defined serum-free conditions. We utilized these growth conditions to examine the mitogenic potential of acidic and basic fibroblast growth factor (aFGF and bFGF) on these cells. Our results demonstrate that both aFGF and bFGF can stimulate the proliferation of keratinocytes and fibroblasts. aFGF is a more potent mitogen than bFGF for keratinocytes. In contrast, bFGF appears to be more potent than aFGF in stimulating the growth of fibroblast cultures. Heparin sulfate (10 micrograms/ml) dramatically inhibited the ability of bFGF to stimulate the proliferation of keratinocytes. In comparison, heparin slightly inhibited the stimulatory effect of aFGF and had no effect on epidermal growth factor (EGF) stimulation in keratinocyte cultures. In fibroblast cultures the addition of heparin enhanced the mitogenic effect of aFGF, had a minimal stimulatory effect on the mitogenic activity of bFGF, and had no effect on EGF-stimulated growth. Our results demonstrate that the proliferation in vitro of two normal cell types found in the skin can be influenced by aFGF and bFGF and demonstrate cell-type specific differences in the responsiveness of fibroblasts and keratinocytes to these growth factors and heparin.     

Isolation of heparin-binding growth factors from bovine, porcine and canine hearts

Fresh bovine, porcine and canine hearts were homogenized and mitogens for mesoderm-derived cells were purified in three different steps. Extraction by two different ammonium sulfate precipitations was followed by cation-exchange chromatography and by heparin-Sepharose affinity chromatography. A heparin-Sepharose fraction from heart (eluted at 1.1 M NaCl) increased mitotic activity in serum-deprived cultures of porcine aortic endothelial and smooth muscle cells, and in human fibroblasts. This mitogenic activity is potentiated by heparin and inhibited by gamma-interferon. The heart mitogenic fraction showed one double peak on HPLC at A215 and one polypeptide band on SDS/PAGE. These peaks and bands were identical to those obtained from bovine brain. The heart acidic fibroblast growth factor (aFGF) showed a positive signal in Western blots using antibodies raised against brain aFGF. Gas-phase amino acid sequencing established that the mitogens were identical to aFGF and the N-terminally truncated aFGF. Extraction in the presence of a protease inhibitor (pepstatin A) produced a higher-molecular mass form of aFGF with a blocked amino terminus. Another mitogen, eluted at 1.6 M NaCl from heparin-Sepharose, reacted with polyclonal antiserum against human recombinant basic fibroblast growth factor (bFGF) and showed a 66% (12 from 18 amino acids determined by gas-phase sequencing) similarity with bFGF. This polypeptide increased the mitotic activity of the same cell lines but was more potent than aFGF.     

Isolation of a receptor for acidic and basic fibroblast growth factor from embryonic chick

A receptor for acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) was isolated from 7-day embryonic chick. Chromatography of solubilized membrane proteins on wheat germ agglutininagarose and aFGF-Sepharose yielded three major polypeptides migrating at 150, 70, and 45 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides were eluted from aFGF-Sepharose with either 1.0 M NaCl or 100 micrograms/ml heparin, but were not retained on underivatized Sepharose. Cross-linking of 125I-aFGF or 125I-bFGF to either crude membrane preparations or to purified fractions yielded a 165-kDa complex, suggesting the existence of a 150-kDa FGF receptor after subtraction of approximately 15 kDa for 125I-FGF. Addition of excess aFGF or bFGF competed for binding of either 125I-aFGF or 125I-bFGF to FGF receptor preparations. Purified FGF receptor fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to Immobilon membranes, and incubated with 125I-aFGF or 125I-bFGF in order to identify FGF-binding polypeptides. Bound 125I-aFGF and 125I-bFGF were displaced by aFGF and bFGF, but not epidermal growth factor, consistent with the identification of the 150-kDa polypeptide as a receptor for acidic and basic FGF. Treatment of purified FGF receptor fractions with N-glycanase demonstrated that the 150-kDa polypeptide contained approximately 10 kDa of N-linked oligosaccharide. The apparent molecular mass of the 150-kDa polypeptide was unaffected by treatment with heparitinase, indicating that the 150-kDa polypeptide is not a heparan sulfate proteoglycan. Together, these data suggest that the 150-kDa polypeptide is a FGF receptor that may mediate the biological activities of aFGF and bFGF.     

Basic and acidic fibroblast growth factors modulate the epidermal growth factor receptor by a protein kinase C-independent pathway

Human acidic and basic fibroblast growth factors (aFGF and bFGF) inhibit epidermal growth factor (EGF) receptor binding in mouse Swiss 3T3 cells. Scatchard analysis indicates that aFGF and bFGF cause a decrease in the high affinity EGF receptor population, similar to that observed for activators of protein kinase C such as phorbol esters, platelet-derived growth factor (PDGF) and bombesin. However, unlike phorbol esters, aFGF and bFGF inhibit EGF binding in protein kinase C-deficient cells. The time course and dose response of inhibition of EGF binding by both aFGF and bFGF are very similar, with an ID50 of approximately 0.10 ng/ml. In contrast to bombesin but like PDGF, neither aFGF nor bFGF act on the EGF receptor through a pertussis toxin-sensitive G protein. These results indicate that both acidic and basic FGF depress high affinity EGF binding in Swiss 3T3 cells with similar potency through a protein kinase C/Gi-independent pathway.     

Cell interactions and regeneration control

This paper is a review of the main findings of our laboratory on the control of regeneration by cell interactions. These include results related to the role of both cell contact and local soluble factors in regeneration of the legs of insects and newts and of the parapodia and segments of nereis. The pattern of these structures is considered to be defined by positional information distributed as longitudinal and transverse positional value sequences carried by epidermal (insect) or mesenchymal (newt) cells. By associating tissues to create transverse and longitudinal discontinuities in these sequences, single or multiple regenerating structures were obtained. These structures are formed by the intercalation of cells characterized by intermediate positional values which fill the gap between the tissues in contact. Positional information may also be changed during regeneration by the nerve cord in nereis and retinoids in the newts. We describe additional cases where morphogenesis occurs without any overt discontinuity in positional information, such as from a locally injured or non-injured insect trochanter, or after deflection of nerves in nereis and newt. Regeneration following an amputation may be considered as a special case of intercalary regeneration, the first stage being the juxtaposition of normally non-contiguous cells resulting in a longitudinal or/and a transverse gap. We also report studies on local factors produced by nerves and the blastema during newt limb regeneration. The nerve factor is necessary for the division of blastemal cells. After denervation, mesenchyme differentiates in an abnormal way. The mitogenic signal from the nerves is mediated by the PKC pathway. Its production is enhanced by regeneration of cut nerve fibers. The blastema also produces growth factors. We show that the epidermal cap and mesenchyme contain acidic FGF-like factor, and that the proliferating mesenchyme stimulates nerve fibers to regrow into the blastema.     

Investigation of the ability of several naturally occurring and synthetic polyanions to bind to and potentiate the biological activity of acidic fibroblast growth factor

The ability of several animal, plant, and bacterial derived polyanions (PAs) as well as synthetic PAs to compete with heparin for the binding of acidic fibroblast growth factor (aFGF) was correlated with their ability to potentiate the mitogenic and neurotrophic actions of this factor. Dextran sulphate, K-carrageenan, pentosan sulphate, polyanethole sulfonate, heparin, and fucoidin competed for the heparin binding site on aFGF at relatively low concentrations (≤50 μg/ml). λ-carrageenan, ι-carrageenan, and polyvinyl sulphate exhibited lower affinity for aFGF, whereas hyaluronic acid, dermatan sulphate, chondroitin-6-sulphate, chondroitin-4-sulphate, and uncharged dextran displayed very low or no demonstrable affinity. Potentiation of the mitogenic action of aFGF for Balb/c 3T3 fibroblasts tended to be in general agreement with the aFGF binding affinity of the PAs. However, polyanethole sulfonate, the carrageenans, polyvinyl sulphate, fucoidin, and pentosan sulphate exerted a mitogenic action on the 3T3 cells that was independent of, and in addition to, the ability of these GAGs to potentiate the action of aFGF. The ability to potentiate the neurotrophic action of aFGF for E8 chick ciliary neurons was a general property of those PA with low or no activity in the mitogen assay. Thus hyaluronic acid, dermatan sulphate, chondroitin-4-sulphate, chondroitin-6-sulphate, and even uncharged dextran all potentiated aFGF induced neuronal survival. The differential effects of these PA in potentiating the biological activities of aFGF are discussed in relation to their ability to compete for the heparin-binding site of aFGF. © 1993 Wiley-Liss, Inc.     

Signal transduction defect appears to be the cause of rat prostate cancer cell fibroblast growth factor insensitivity.

Using monolayer cultures of clonally isolated C3 and T5 rat prostate cancer cells, we determined that acidic (aFGF) and basic (bFGF) fibroblast growth factors profoundly enhanced T5 cell thymidine incorporation with half-maximum stimulation at 0.53 and 0.35 ng/ml, respectively. In contrast, aFGF or bFGF enhancement of C3 cell thymidine incorporation was about 5% of that of T5 cells, and effects were principally mitogen concentration independent. Saturation analyses and cross-linking studies established that both C3 and T5 cells contained high-affinity FGF receptors of 120 and 145 kilodaltons and that receptor content and Kd of C3 and T5 cells were comparable. aFGF or bFGF stimulation of T5 cell thymidine incorporation profoundly decreased as cell plating density was reduced from 1.5 x 10(5) to 1.0 x 10(4) cells/well. The modest response of C3 cells to either aFGF or bFGF also decreased as cell plating density was reduced. Because heparin preserves FGF biological activity and enhances bFGF binding to high-affinity FGF receptors, we examined the effect of heparin on FGF stimulation of C3 cell thymidine incorporation. We found that changes in cell plating density and/or medium heparin concentration had variable, inconsistent effects. These were C3 cell plating density associated and included inhibition or modest enhancement of FGF effects. Binding analyses established that high-affinity bFGF binding of C3 and T5 cells immediately prior to assessing FGF-stimulated thymidine incorporation was comparable and independent of cell plating density, implying that C3 cell FGF insensitivity was not attributable to differences in C3 and T5 cell FGF receptor content at the time of mitogen stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heparan sulfate proteoglycan modulates keratinocyte growth factor signaling through interaction with both ligand and receptor

Keratinocyte growth factor (KGF) is an unusual fibroblast growth factor (FGF) family member in that its activity is largely restricted to epithelial cells, and added heparin/heparan sulfate inhibits its activity in most cell types. The effects of heparan sulfate proteoglycan (HSPG) on binding and signaling by acidic FGF (aFGF) and KGF via the KGFR were studied using surface-bound and soluble receptor isoforms expressed in wild type and mutant Chinese hamster ovary (CHO) cells lacking HSPG. Low concentrations of added heparin (1 microgram/mL) enhanced the affinity of ligand binding to surface-bound KGFR in CHO mutants, as well as ligand-stimulated MAP kinase activation and c-fos induction, but had little effect on binding or signaling in wild type CHO cells. Higher heparin concentrations inhibited KGF, but not aFGF, binding and signaling. In addition to the known interaction between HSPG and KGF, we found that the KGFR also bound heparin. The biphasic effect of heparin on KGF, but not aFGF, binding and signaling suggests that occupancy of the HSPG binding site on the KGFR may specifically inhibit KGF signaling. In contrast to events on the cell surface, added heparin was not required for high-affinity soluble KGF-KGFR interaction. These results suggest that high-affinity ligand binding is an intrinsic property of the receptor, and that the difference between the HSPG-dependent ligand binding to receptor on cell surfaces and the HSPG-independent binding to soluble receptor may be due to other molecule(s) present on cell surfaces.     

Isolation of heparin-binding growth factors from dogfish (Mustela canis) brain and retina

We have purified two growth factors from dogfish brain and retina by using their binding affinity for heparin-Sepharose. These growth factors were eluted at 1 and 2 M NaCl similarly to those purified from bovine brain or retina. Their mitogenic activity was assayed in vitro on the same mammalian cells: bovine lens epithelial cells or human fibroblasts. All these data seem to indicate that these growth factors belong to the families of other well defined mammalian growth factors: EDGF I, brain basic FGF, AGF II, on the one hand and EDGF II, brain acidic FGF, AGF I, ECGF, on the other. Thus, these growth factors have been widely distributed during evolution and retain at least a conservative sequence to stimulate cell proliferation, in mammalian cells.