Heparan sulfate potentiates the autocrine action of basic fibroblast growth factor in astrocytes: an in vivo and in vitro study

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Neuroscience Vol. 76, No. 1, pp. 137–145, 1997 Copyright ? 1996 IBRO. Published by Elsevier Science Ltd Printed in Great Britain 0306–4522/97 $17.00+0.00 S0306-4522(96)00327-2

HEPARAN SULFATE POTENTIATES THE AUTOCRINE ACTION OF BASIC FIBROBLAST GROWTH FACTOR IN ASTROCYTES: AN IN VIVO AND IN VITRO STUDY F. GO u MEZ-PINILLA*, S. MILLER, J. CHOI and C. W. COTMAN Institute for Brain Aging and Dementia, and Department of Neurology, University of California, Irvine, CA 92697-4540, U.S.A. Abstract––Increasing evidence indicates that heparan sulfate proteoglycans have a critical role in the regulation of the activity of basic fibroblast growth factor by interacting with it or its receptor. In this study we examined the possibility that heparan sulfate can modulate the basic fibroblast growth factor system at a more fundamental level than activity regulation, by influencing the synthesis of basic fibroblast growth factor and its receptor messenger RNAs. Previous studies in vitro indicate that basic fibroblast growth factor promotes proliferation and differentiation of astrocytes. Accordingly, we examined the possibility that the action of heparan sulfate on the basic fibroblast growth factor system could have a critical role in the modulation of reactivity and/or proliferation of astrocytes in vitro and in vivo. We report that basic fibroblast growth factor applied to pure astrocyte cultures or rat neocortex promoted an increase in the messenger RNA for basic fibroblast growth factor itself and for its receptor. Furthermore, basic fibroblast growth factor applied directly into the brain elicited an increase in messenger RNA for the astrocytic marker glial fibrillary acidic protein. All of these actions, both in vitro and in vivo, were highly potentiated when heparan sulfate was applied in combination with basic fibroblast growth factor. These results suggest that basic fibroblast growth factor regulates astrocytic proliferation or reactivity via an autocrine cascade that involves induction of its own receptor, and that this action is modulated by heparan sulfate. Copyright ? 1996 IBRO. Published by Elsevier Science Ltd. Key words: FGF-2, FGF receptor, plasticity, trauma, growth factors.

The interactions between growth factors and the extracellular matrix (ECM) are critical for the development and regeneration of the nervous system.9,27,28,31,46,50 Accordingly, it is important to understand the nature of these interactions. Increasing evidence suggests that ECM proteoglycans have a key role in neural and glial migration and guidance of process outgrowth in the developing CNS14,24,28,31,55 and in adult plasticity.40 In turn, basic fibroblast growth factor (FGF-2), the most extensively studied member of the fibroblast growth factor (FGF) family (see Baird4 for review), has important actions in the development and regeneration of the brain. FGF-2 has a characteristic broad spectrum of activity on glial43 and neuronal cells,1,58 and its expression is developmentally regulated.6,19,23,4460 Furthermore, FGF-2 is involved in brain repair following traumatic injury13,17,18,36 and appears to have a role in *To whom correspondence should be addressed. Abbreviations: DMEM, Dulbecco’s modified Eagles’ medium; ECM, extracellular matrix; EDTA, ethylenediaminetetra-acetate; FCS, fetal calf serum; FGF, fibroblast growth factor; FGF-1, acidic fibroblast growth factor; FGF-2, basic fibroblast growth factor; FGFR, fibroblast growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; HEPES, N-2-hydroxyethylpiperazine-N*2-ethanesulfonic acid; HS, heparan sulfate; PIPES, (Piperazine-N,N*-bis[2-ethanesulfonic acid).

plaque biogenesis in Alzheimer’s disease.16 The versatility of FGF-2 suggests that it must be tightly regulated in order to maintain brain homeostasis. One of the most important modulators of FGF-2 action is heparin and related molecules. In fact, FGFs are typically characterized by their affinity for heparan sulfate (HS) and related molecules, which has aided in their isolation and purification and led to the synonym ‘‘heparin binding growth factors’’.21,35 Extensive studies have demonstrated that, in vitro, the functioning of the FGF-2 system can be modified by interaction with HS (see Mason37 for review). Specifically, HS or related molecules can bind independently to FGF-210,22,59 and/or its receptor,25 modifying FGF-2 stability, receptor binding and function.3,27,46,61 In fact, when FGF-2 is complexed with HS, it is internalized,49 it is protected from heat and proteolytic inactivation,51 and it is more potent than FGF-2 alone.3 HS molecules are generally attached to a protein core, forming an HS proteoglycan molecular complex. HS constitutes most of the mass of the complex and appears to be largely responsible for its biological activity.24,28 Proteoglycans constitute up to 90% of the ECM volume, and are found on the surfaces of all adherent cells and within intracellular vesicles.28 Various HS proteoglycans that have important roles in development and plasticity of the CNS have recently been identified in the brain.28,31

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Interestingly, emerging evidence indicates that the expression and function of proteoglycans in the CNS are more malleable than thought previously and, like FGF-2, appear to be regulated by injury29,30,32 and neural activity.40 Thus, proteoglycans acting in conjunction with growth factors may have critical roles in development and plasticity of the CNS. FGF-2 is primarily expressed in astrocytes and participates in their differentiation33,39 and proliferation.15,43 Evidence showing that FGF-2 promotes the synthesis of its own mRNA in cultured astrocytes supports the idea that the action of FGF-2 on astrocytes appears to be achieved in an autocrine fashion. If an autocrine mechanism is involved in FGF-2 regulation in astrocytes, this process would require modulation of its receptor (FGFR). Furthermore, based on current evidence, it is likely that modulation of the action of FGF-2 in astrocytes involves an interaction with HS proteoglycans. In spite of the large number of studies on the regulation of the activity of FGF-2, interactions between FGF-2 and HS in influencing gene expression have not been examined.

EXPERIMENTAL PROCEDURES

Cell culture Primary glial cultures were prepared from neocortices of neonatal rat pups (three to four days old) using a modification of the method of McCarthy and de Vellis.38 Briefly, neocortex was dissected, dissociated by trituration, and plated on poly--lysine-precoated flasks in Dulbecco’s modified Eagles’ medium (DMEM, pH 7.4) buffered with 25 mM HEPES and 14.3 mM NaHCO3 and supplemented with 15% fetal calf serum (FCS), 1 mM pyruvate and 2 mM glutamine. For subsequent feeding, the FCS supplementation was reduced to 10%. After five to six days, the flasks were shaken overnight (280 r.p.m.) to remove oligodendrocytes and microglia. One day following this purification step, secondary astrocyte cultures were established by trypsinizing and subplating into poly--lysine-precoated 100-mm culture dishes in DMEM + 10% FCS at a density of 106 cells/dish. After three days in secondary culture, the astrocytes were rinsed and switched to a minimally supplemented serum-free control medium: DMEM with transferrin (50 µg/ml), -biotin (10 ng/ml), selenium (5.2 ng/ml), fibronectin (1.5 µg/ml) and insulin (5 µg/ml). For experimental conditions HS (0.5 µg/ml; sodium salt, bovine kidney, mol. wt c. 12,000; Sigma) and/or human recombinant FGF-2 (5 ng/ml; UBI, NY, U.S.A.) were included in the serum-free medium for 48 h before the cells were harvested for RNA extraction. Surgical procedures Sprague–Dawley male rats (Charles River, NY, U.S.A.), three to four months old, received bilateral aspirations of a small segment of the sensorimotor cortex (1.5 mm diameter, 1.7 mm ventral from the top of the skull, 3.5 mm lateral from the midline). One group of rats (n = 4) received bilaterally in the wound cavity a piece of gelfoam embedded with: recombinant FGF-2 (100 ng/ml; UBI, NY), HS (10 µg/ml; sodium salt, bovine kidney, mol. wt c. 12,000; Sigma) or both, dissolved in phosphate-buffered saline. A group of rats to be used as a control received a piece of gelfoam soaked with phosphate-buffered saline bilaterally in

the neocortex. After 48 h, rats were quickly decapitated, and the neocortex around the wound cavity (0.5 cm thick) was dissected out and frozen in dry ice. Nuclease protection assay Total cellular RNA from culture cells or brain tissue was isolated by guanidine thiocyanate extraction according to Chomczynski and Sacchi.8 Total RNA (20 µg) was dissolved in 30 µl of hybridization buffer (80% formamide, 40 mM PIPES, pH 6.4, 400 mM NaCl and 1 mM EDTA) containing 5 µl of a 32P-labeled cRNA probe (106 c.p.m.). After heating at 85)C for 10 min to denature RNA, the cRNA probe was allowed to anneal the endogenous mRNA at 55)C overnight. At the end of the hybridization, the solution was diluted with RNase digestion buffer containing 40 µg/ml of RNase A and 2 µg/ml of RNase T1, and incubated for 30 min at 37)C. Following proteinase K (240 µg/ml) digestion, RNA was extracted with phenol– chloroform and precipitated with ethanol. The pellet containing the RNA–RNA hybrid was resuspended in gelloading buffer, boiled at 85)C and separated on a 5% polyacrylamide, 7 M urea gel unit. The gel was dried and the protected fragment was visualized by autoradiography on âmax hyperfilm (Amersham, Illinois, U.S.A.). In some experiments a separate hybridization was performed with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cRNA probe to assure that samples used for the assay contained an equivalent amount of RNA. Relative levels of mRNA were estimated by measuring the peak densitometry area of the autoradiogram using an image analysis system (Imaging Inc., St Catherines, Ontario, Canada). Data are expressed as percentage of control. The FGFR-1 riboprobe was prepared from cDNA templates containing a unique sequence encoding the extracellular and transmembrane domain of the human FGFR-1 gene60 (kindly provided by Dr Jeffrey Mildbrandt). This fragment did not overlap the tyrosine kinase domain. A pBluescript of approximately 350 bp cDNA, containing a fragment of approximately 300 bp encoding the FGFR-1 gene (protected fragment in the nuclease protection assay), was linearized with EcoRI and transcribed with T3 RNA polymerase. The FGF-2 riboprobe was prepared from cDNA templates encoding a unique sequence of the rat FGF-2 gene56 (clone RObFGF503; kindly provided by Dr Andrew Baird). A pBluescript SK+ plasmid was linearized with NcoI and then transcribed using T7 RNA polymerase to generate a 524-base anti-sense strand which contained a 477-base FGF-2 gene-coding region (protected fragment in the nuclease protection assay). To assess relative levels of the mRNA for the astrocytic marker glial fibrillary acidic protein (GFAP), nuclease protection assay was performed using GFAP cRNA templates encoding the rat GFAP gene (kindly provided by Dr Caleb Finch). A 364-base cRNA probe that contained a 309-base (nuclease protection assay-protected fragment) unique sequence encoding the GFAP gene was linearized with NsiI and transcribed with T3 RNA polymerase. As a control, a 376-base GAPDH cRNA probe was generated by transcribing a manufacturer pre-linearized plasmid (Ambion, Texas, U.S.A.) with T3 RNA polymerase. Transcription reactions were performed using a Promega transcription kit (Promega, Wisconsin, U.S.A.) and (á-32P)CTP (Amersham; 800 Ci/mmol). RESULTS

FGF-2 up-regulated FGF receptor-1 and FGF-2 mRNAs in astrocytes in vitro Previous evidence indicates that FGF-2 is mitogenic for astrocytes, and this action may follow

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Fig. 1. Changes in FGFR-1 mRNA levels following HS, FGF-2 or FGF-2/HS treatments were assessed in cultures of purified secondary astrocytes. Astrocytes were incubated with HS (0.5 µg/ml), FGF-2 (5 ng/ml), FGF-2/HS or control media for 48 h and then harvested for a nuclease protection assay. (A) The graph shows a relative quantification obtained by computer densitometric analysis of autoradiograms and expressed as a percentage of control. There were significant increases in FGFR-1 mRNA following all the treatments as compared to control and across the different conditions. (B) The leftmost lane of the sample gel displays the approximately 350-base 32P-labeled anti-sense cRNA probe and the adjacent lanes display the protected FGFR-1 mRNA fragment (approximately 300 bases). Size markers are indicated at the far right as a reference. At the bottom of B is shown the expression of a GAPDH cRNA probe used as a control for loading equal amounts of total RNA for each condition. Values shown are the means & S.D. of three experiments. *P < 0.05, **P < 0 01. Statistical comparisons were performed using ANOVA and Fisher’s test.

an autocrine mechanism of activation.11 Accordingly, we examined the possibility that FGF-2 has an active role on the induction of the mRNA for its receptor and/or itself in cultured astrocytes from neonatal rat neocortex. A nuclease protection assay revealed that FGF-2 treatment elicited a 50% increase in FGFR-1 mRNA (P < 0.01; Fig. 1) and a 35% increase in its own mRNA (P < 0.01; Fig. 2), compared with astrocytic cultures that were maintained in control medium. The present evidence indicates that FGF-2 regulates its own mRNA together with that of its receptor mRNA in cultured astrocytes. Heparan sulfate potentiated the effect of FGF-2 in astrocytes in vitro Evidence suggests that binding of HS either to FGFR25 or FGF-2 itself3,27,46,61 appears to be critical for the modulation of the action of FGF-2. Here, we examined the possibility that HS influences the synthesis of FGFR or FGF-2 mRNAs, in essentially pure secondary cultures of astrocytes. Treatment of cultures with HS alone triggered a 15% increase in FGFR-1 mRNA (P < 0.05; Fig. 1) and a 20% increase in FGF-2 mRNA (P < 0.05; Fig. 2). Interestingly, the combined application of FGF-2 and HS highly potentiated the effects of either FGF-2 or HS alone, triggering a three-fold increase in the levels of FGFR-1 mRNA (P < 0.01;

Fig. 1) and a 3.5-fold increase in FGF-2 mRNA (P < 0.01; Fig. 2). There were some morphological changes associated with the different treatments (Fig. 3). Cells cultured in the control medium had a flat, confluent appearance with few processes (Fig. 3A), while cells exposed to HS showed a small increase in the number and length of processes (Fig. 3B). Exposure to FGF-2 produced an increase in cell density and process elaboration as compared to cells exposed to HS (Fig. 3C), while cells exposed to a combination of both HS and FGF-2 showed a further increase in density and stellation; processes were longer and finer than cells exposed to any of the previous treatments (Fig. 3D). Our results indicate that FGF-2 regulates its receptor mRNA and its own mRNA in cultured astrocytes, and that these actions are modulated by HS. To investigate the possibility that the results obtained from in vitro studies also apply in vivo, we examined the effects of similar treatments in the brain. Our paradigm in vivo was based on the fact that FGF-2 and its receptor are up-regulated in response to injury17,18,36 in the brain, and on the possibility that their levels could be maximized by increasing the availability of FGF-2 and/or HS to cells. Accordingly, a group of rats received aspiration of a small segment of the sensorimotor cortex, and then a piece of gelfoam embedded with recombinant FGF-2, HS, FGF-2 combined with HS, or saline was applied in the wound cavity.

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when compared to the treatment with FGF-2 alone. These results in vivo are parallel to the results in astrocytic cultures, and indicate that FGF-2 regulates the mRNAs of its receptor and of itself in the brain, and that these actions are potentiated by HS.

FGF-2 and heparan sulfate up-regulated GFAP mRNA in the neocortex in vivo

Fig. 2. Changes in FGF-2 mRNA expression following HS, FGF-2 or FGF-2/HS treatments were assessed in astrocytic cultures using a nuclease protection assay. Astrocytes were incubated for 48 h with HS (0.5 µg/ml), FGF-2 (5 ng/ml), FGF-2/HS or control media. As the graph illustrates, there were significant changes in FGF-2 mRNA following all the treatments as compared to control (C) and across the different conditions. The inset sample gel illustrates the typical pattern of the 524-base 32P-labeled anti-sense cRNA probe, which contained a 477-base FGF-2 gene-coding region, used to assess relative levels of FGF-2 mRNA. The leftmost lane of the gel displays the FGF-2 cRNA probe (524 bases) and the adjacent lanes display the protected FGF-2 mRNA fragment (477 bases). GAPDH controls are shown in Fig. 1. Size markers are indicated at the far right as a reference. Values shown are the means & S.D. of three experiments. *P < 0.05, **P < 0.01. Statistical comparisons were performed using ANOVA and Fisher’s test.

Experimental evidence from studies in vitro indicates that FGF-2 can promote the proliferation of astrocytes.43 Moreover, our experiments in cultured astrocytes showed an increase of FGF-2 and FGFR-1 mRNAs following FGF-2 and HS applications. Therefore, we examined the possibility that the results of our experiments, in vitro and in vivo, could be related to astrocytic proliferation or reactivity. Accordingly, FGF-2, HS or FGF-2/HS were applied into the neocortex and total RNAs were evaluated for the mRNA of the astrocytic marker GFAP using a nuclease protection assay. The results showed that FGF-2 treatment elicited a 35% increase (P < 0.01; Fig. 6) in GFAP mRNA in the area surrounding the lesion cavity. HS applied alone triggered a 10% increase (P < 0.05) in GFAP mRNA. The effect of FGF-2 treatment on GFAP induction was highly potentiated by the combined application of FGF-2 and HS, triggering a 70% increase in GFAP mRNA (P < 0.01; Fig. 6). These results show that FGF-2 induces proliferation and/or reactivity of astrocytes in the brain, and that this action is regulated by HS.

FGF-2 up-regulated FGF receptor-1 and FGF-2 mRNAs in the neocortex in vivo Rats that were treated with recombinant FGF-2 after the lesion showed a 20% increase in FGFR-1 mRNA (P < 0.01; Fig. 4) and a 25% increase in FGF-2 mRNA (P < 0.01; Fig. 5), as compared to rats that received saline only. The results of these experiments in vivo indicate that, similar to previous studies in vitro, FGF-2 induces FGFR-1 mRNA and its own mRNA. Heparan sulfate potentiated the effect of FGF-2 in the neocortex in vivo We examined the possibility that HS influences the synthesis of FGFR-1 mRNA and/or FGF-2 mRNA in the brain. The application of HS into a wound cavity did not show any detectable effect on FGFR-1 mRNA (Fig. 4) or FGF-2 mRNA in the surrounding cells (Fig. 5). Interestingly, the combined application of FGF-2 and HS triggered a 45% increase in FGFR-1 mRNA (P < 0.01; Fig. 4) and a 50% increase in FGF-2 mRNA (P < 0.01; Fig. 5), as compared to saline control rats. The increases in FGFR-1 mRNA (P < 0.01) and FGF-2 mRNA (P < 0.01) were also significantly greater

DISCUSSION

Our results show that FGF-2 regulates the synthesis of the mRNA for its receptor and for itself both in vitro and in vivo, and these actions are highly potentiated by HS. It is likely that these events are associated with the regulation of astrocyte proliferation and/or reactivity, since the changes in FGFR-1 and FGF-2 mRNAs were prevalent in cultured astrocytes and closely correlated with changes in GFAP mRNA in the brain. Furthermore, our recent work indicates that FGF-2 and HS also affect the phenotypic expression of FGF-2 and FGFR-1 proteins in astrocytes in vivo.20 The present evidence suggests that the action of FGF-2 in astrocytes may involve an autocrine cascade in which FGFR-1 and FGF-2 mRNAs are induced under HS proteoglycan regulation. FGFR and its mRNA are present at low levels in the intact brain;18,36,60 however, they are highly regulated by stimuli such as trauma18,36 or physiological activity.48,57 The present data suggest that the levels of FGFR-1 and FGF-2 induced by injury are probably sub-maximal and can be enhanced by exogenous FGF-2 and/or HS applications. Therefore, the

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Fig. 3. The phase-contrast microphotograph shows the typical phenotype of culture cells associated with the HS, FGF-2 and FGF-2/HS treatments. Astrocytes cultures were incubated with HS (0.5 µg/ml), FGF-2 (5 ng/ml), FGF-2/HS or control media for 48 h. (A) Cells incubated in control media had a flat, confluent morphology with scarce and short processes (arrows). (B) Cells treated with HS showed a small increase in stellation, with greater process elaboration (arrows). (C) FGF-2 treatment produced a considerable increase in cell density and process formation (arrows). (D) Treatment with a combination of FGF-2 and HS further increased process formation and extension (arrows), and produced a small increase in density compared with cultures treated with FGF-2 alone. Scale bar = 10 µm.

events described here, both in vivo and in vitro, may be highly relevant for brain plasticity. Heparan sulfate interacts with FGF-2 and FGF receptor Current evidence indicates that HS proteoglycans have a role in the regulation of the action of FGF-2 on cells; however, the mechanism involved remains unclear. FGFs possess a high affinity for HS or related molecules, and this property has been a key factor in its purification from tissue.21,35 In fact, studies have described that HS is required for binding of FGF-2 to its signal transduction receptor, and prevention of this binding blocks the biological activity of FGF-2.3,27,46,61 Interestingly, our data suggest that the presence of HS influences the gene expression of FGFR-1 and FGF-2. However, our results showing that HS treatment triggers an increase in FGF-2 and FGFR-1 mRNAs do not exclude the possibility that HS may act in combination with FGF-2 from endogenous sources. Furthermore, changes in mRNA levels may be partially influenced by changes in mRNA transcription rate and/or

mRNA stability. It is noteworthy that the potentiating effect of HS on the ability of FGF-2 to induce FGF-2 and FGFR-1 mRNAs is not simply additive, suggesting its importance in the regulation of FGF-2 function. Kan et al.25 have recently shown in vitro that HS interacts with a specific region in the extracellular domain of the FGFR itself, at a site distinct from FGF-2. It is likely, therefore, that the effects of HS on FGFR-1 gene expression may be elicited in part by direct binding to FGFR-1. This hypothesis differs from previous hypotheses maintaining that any effect of HS on FGF-2 function would be achieved exclusively by forming a complex with FGF-2. These results further suggest that modulation of the FGFR-1 may be a factor in the control of the action of FGF-2. Heparan sulfate proteoglycans act in concert with FGF-2 in the brain Present evidence suggests that a proper balance between FGF-2 and HS is relevant to control the action of FGF-2 in the brain. Indeed, recent studies

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Fig. 4. Changes in FGFR-1 mRNA levels in the rat brain elicited by treatments with HS, FGF-2 or FGF-2/HS. A wound cavity in the neocortex was filled with a piece of gelfoam embedded with saline, HS (10 µg/ml), FGF-2 (100 ng/ml) or FGF-2/HS, and the tissue surrounding the wound cavity was assayed with a nuclease protection assay. (A) As shown in the graph, there were significant changes in FGFR-1 mRNA following FGF-2 and FGF-2/HS treatments as compared to saline control and HS, and between FGF-2 and FGF-2/HS. (B) The sample gel illustrates the typical pattern of the FGFR-1 mRNA response obtained using the 350-base 32P-labeled anti-sense cRNA probe. The leftmost lane of the gel displays the FGFR-1 cRNA probe (approximately 350 bases) and the other lanes display the protected FGFR-1 cRNA fragment (approximately 300 bases). Size markers are indicated at the far right as a reference. Shown at the bottom of B is the expression of a GAPDH cRNA probe used to control for loading equal amounts of total RNA for each condition. Values shown are the means & S.D. of four experiments. * P < 0.05, **P < 0.01. Statistical comparisons were performed using ANOVA and Fisher’s test.

suggest that HS proteoglycans may act as direct transducers of FGF-2 signaling.45 HS proteoglycans, as well as other ECM components, show different expression patterns14,24,28,55 and different biological activities28,31 throughout ontogeny of the brain. For example, Nurcombe et al.42 recently reported that differences in the type of HS carried by a single core protein can switch the affinity for FGF-1 or FGF-2, and that these changes are developmentally regulated. In addition, the structures and biological activities of HS proteoglycans may vary across different brain regions.12,14 Several HS proteoglycan subtypes have recently been identified that have important roles in development and plasticity of the CNS.28,31 However, it is not clear which of them is primarily responsible for the regulation of FGF-2 activity in vivo. Studies, mainly based on the affinity of FGF– proteoglycan binding, postulate that syndecans, cloned by Bernfield’s group,52 are low-affinity FGFRs.26,27,50,52 However, recent studies have found that other HS proteoglycans, for example glypican,5,7,34 are important regulators of FGF-2’s action on cells as well. Aviezer et al.2 have recently shown that the basal lamina HS proteoglycan perlecan41,53 is a major activator of FGF-2, promoting highaffinity binding in vitro and angiogenesis in vivo. Similar to FGF-2, the expression and function of proteoglycans in the CNS is malleable,29,30,32,40 suggesting that the combined action of proteoglycans

and FGF-2 may have a critical role in the development and plasticity of the CNS.

The interaction between heparan sulfate and FGF-2 is significant for astrocytic physiology Our results that FGF-2 induces FGF-2 and FGFR-1 mRNAs in highly pure astrocytic cultures support and complement our results in vivo, showing that FGF-2 induces GFAP mRNA, and suggest that FGF-2 may stimulate astrocytic proliferation and/or reactivity.15,43 Furthermore, these results support the hypothesis that the action of FGF-2 in astrocytes may follow an autocrine mechanism. According to this view, FGF-2 induction of FGFR-1 and FGF-2 mRNAs would enhance the ability of astrocytes to respond to FGF-2, thus potentiating astrocytosis. Previous studies in vitro have shown that the action of FGF-2 on astrocytes is diverse and includes modulation of differentiation,33,39 proliferation,11,15,43 migration54 and regulation of other trophic molecules.62 The action of select brain proteoglycans on FGF-2 or FGFR-1 in astrocytes may provide a fine mechanism by which the action of FGF-2 is co-ordinated with particular physiological demands. A large body of evidence indicates that FGF-2 is also critical to neuronal function.1,4,58 Interestingly, recent studies have shown that FGF-2 may induce either survival or

Extracellular matrix modulation of FGF mRNA

Fig. 5. Changes in FGF-2 mRNA levels in the brain elicited by applications of HS, FGF-2 or FGF-2/HS. A wound cavity in the neocortex was filled with a piece of gelfoam embedded with saline, HS (10 µg/ml), FGF-2 (100 ng/ml) or FGF-2/HS, and the tissue surrounding the wound cavity was assayed with a nuclease protection assay. As shown in the graph, there were changes in FGF-2 mRNA following FGF-2 and FGF-2/HS treatments as compared to control (C) and HS, and between FGF-2 and FGF-2/HS. A 524base 32P-labeled anti-sense cRNA probe which contained a 477-base FGF-2 gene-coding region was used in a nuclease protection assay. The sample gel displays the FGF-2 cRNA probe (leftmost lane, 524 bases) and the other lanes display the protected FGFR-1 cRNA fragment (477 bases). Size markers are indicated at the far right as a reference. GAPDH controls are shown in Fig. 4B. Values shown are the means & S.D. of four experiments. *P < 0.05, ** P < 0.01. Statistical comparisons were performed using ANOVA and Fisher’s test.

proliferation of embryonic progenitor neurons in vitro in a dose-dependent manner.47 CONCLUSIONS

The present evidence indicates that FGF-2 regulates the synthesis of the mRNA for its receptor and for itself, and that these actions are highly potentiated by HS. It is likely that these events are associated with the regulation of astrocyte proliferation and/or reactivity involving an autocrine cascade under HS proteoglycan regulation. Therefore, the FGFR-1/FGF-2 system, via interaction with ECM

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Fig. 6. Changes in GFAP mRNA levels concomitant with changes of FGFR-1 mRNA and/or FGF-2 mRNA levels were assayed in the neocortical tissue adjacent to applications of HS, FGF-2 or FGF-2/HS. A wound cavity in the neocortex was filled with a piece of gelfoam embedded with saline, HS (10 µg/ml), FGF-2 (100 ng/ml) or FGF-2/HS, and the tissue surrounding the wound cavity was assayed with a nuclease protection assay. As illustrated in the graph, there were significant changes in GFAP mRNA levels following all the conditions as compared to controls, and across the different treatments. A 364-base 32P-labeled anti-sense cRNA probe, which contained a 309-base GFAP gene-coding region was used to assess relative levels of GFAP mRNA with a nuclease protection assay. The leftmost lane of the sample gel displays the GFAP cRNA probe (364 bases) and the adjacent lanes display the protected GFAP mRNA fragment (309 bases). Size markers are indicated at the far right as a reference. GAPDH controls are illustrated in Fig. 4B. Values shown are the means & S.D. of four experiments. *P < 0.05, **P < 0.01. Statistical comparisons were performed using ANOVA and Fisher’s test.

components and probably other factors, may be involved in a cascade of events that regulates development and plasticity of astrocytes, and probably neuronal cells in the CNS. Acknowledgements—This work was supported by the American Paralysis Association award GAR 1-9504 and NIA Program Project AG07918. We thank Dr Jennifer Kahle for critical review of this manuscript.

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