Dez'eiopmental Brahz Research, 68 ( ! 992) 247-253 ct~ 1992 Elsevier Science Publishers B.V. All rights reserved 0165-3806/92/$05.110

247

BRESD 51494

Embryonic brain-derived heparan sulfate inhibits cellular membrane binding and biological activity of basic fibroblast growth factor H u b e r t H o n d e r m a r c k a, E l i s a b e t h D e u d o n b a n d B 6 n o n i Boilly

a

a Laboratoire de Biologic des Facteurs de Croissance, Unicersitt; des Sciences et Techniques de Lille, Villem.we d'Ascq (France) and t, Laboratoire de Biochimie, INSERM UI81, Facultt; de M~decine Saint Antoine, Paris (France)

(Accepted 5 May 1992)

Key words: Heparan sulfate; Fibroblast growth factor; ~.~,nbryonicbrain; Chick

We have investigated the ,~bility of glycosaminoglycans from embryonic chick brain (15 days old~ to interact with basic fibroblast growth factor (bFGF). 35SO4 metabolic,lily labeled glycosaminoglycans were purified and separated on DEAE-cellulose chromatography. Material which eluted between 0.20 and 0.:35 M NaCI displaced the binding of [ t-'Sl]bFGF to brain membrane. This activity was dose-dependent and on the basis to its heparinase sensitivity and chondroitinase insensitivity, has been attributed to heparan sulfate. CL-6B-Sepharose chromatography of this material revealed two glycosaminoglycans of molecular masses of about 15,000 and 65,000. Incubation with [1251]bFGF followed or not by heparinase and chondroitinase treatment of electrotransfert from SDS-PAGE revealed that both of these forms correspond to heparan sulfate chains and bind bFGF. in vitro, embryonic brain-derived heparan sulfate inhibited both bFGF induced [3H]thymidine incorporation in CCL39 cells and neurite outgrowth in PCI2 cells. These results suggest that heparan sulfate play an important function in the control of whe biological activity of bFGF during brain development.

INTRODUCTION Fibroblast growth factors are a family of structurally related polypeptidcs including the prototypes acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), int-2, hst, FGF5, k.FGF and KGF. In vitro, the biological effects of these growth factors are widespread. They affect morphology, proliferation, migration, differentiation and senescence of various types of mesoderm- and neuroectoderm-derived cells 2. The biological response to FGFs is mediated through specific high affinity (dissociation constant (K o) = 10 -~°10 -t2 M) cell membrane receptors. A lower affinity (K d = 10-8-10 -9 M) category of binding sites has also been identified and correspond to heparin-like molecules found on the cell surface and in the extracellular matrix. The interaction with heparin-like structures protect FGFs from denaturation and enzymatic inactivation t~,37 and thus allows the storage of this growth factor in the extracellular matrix 44. Therefore, it has recently been demonstrated that heparin-like

molecules are required for the binding of bFGF to its high affinity receptors; so these binding sites could be directly involved in bFGF cell signaling 20,32,48. However, most information concerning the effects of FGFs have been obtained in vitro and little is known about the mechanism of action of tt:ese growth factocs in vivo. Since FGFs have both mitogenic and neurotrophic properties and are found in important quantities in the nervous,tissue, brain development has been studied as a model of in vivo FGFs activity. Thus, aFGF and bFGF have been purified from rat 5, mouse 4, and chick 27.35embryonic brain. An immunohistochemical study indicated that bFGF is widely distributed in the brain but more concentrated in the high density neurons areas, in endothelial cells and in extracellular matrix 10. FGF mRNA have been detected in mouse 4~ and chick 39 embryonic brain. In situ hybridization experiments indicated the presence of FGFs mRNA in developing and mature n e u r o n s 39. Both the quantity of FGFs 5,34,41 and of their mRNA 41 have been shown to increase during brain development. As

Correspondence: H. Hondermarck. Present address: Department of Biological Chemistry, College of Medicine, University of California, Irvine, CA 92717, USA. Fax: (1) (714) 725-2688.

248 examp le, in chick b r a i n , t h e F G F biological activity increases from day 6 to d a y 14 of e m b r y o g e n e s i s a n d t h e n r e m a i n s relatively stable until day 18 34. I n t e r e s t ingly, we d e m o n s t r a t e d t h a t the q u a n t i t y o f high affinity r e c e p t o r s for F G F s d e c r e a s e s b e t w e e n d a y 7 a n d d a y 10 of embryonic chick b r a i n d e v e l o p m e n t ~s. In t h e s a m e report, we s h o w e d t h a t the n u m b e r o f low affinity m e m b r a n e binding sites for F G F s i n c r e a s e s d u r i n g d e v e l o p m e n t a n d especially b e t w e e n day 15 o f e m b r y onic d e v e l o p m e n t a n d birth. T h e s e d a t a suggest, as shown in vitro .~:,4s, t h a t t h e interaction of F G F with its high affinity receptors, d u r i n g b r a i n d e v e l o p m e n t , could be r e g u l a t e d by the low affinity binding sites, i.e. heparin-like molecules. In the p r e s e n t report, we f o c u s e d on the function of the low affinity F G F s b i n d i n g sites in embryonic brain. F o r this p u r p o s e , we p u r i f i e d hepa r a n sulfate molecules f r o m e m b r y o n i c chick b r a i n a n d studied their ability to bind b F G F a n d to m o d i f y both the cellular m e m b r a n e b i n d i n g a n d the biological activities of this growth factor. MATERIALS A N D M E T H O D S

Materials Embryonic chick brains were obtained from White Leghorn eggs at 15 days of development. Care wa:~ taken to avoid meningeal ¢ontaminatkm. bFGF was purified to homogeneity from bovine brain as described ~ and iodinated using the chloramine-T method as

previousl~ reported ~".

/~'~SO~] metabolic hlheling o[ brains Fifty embryonic brains were dissected, lightl~ homogeneiz~d in a glass homo~,~nizer and immediately incubated ivl :rl ml of phosphate buffer, 50 raM, pit 7.4 containing 50 ~Ci/ml of No'X:~)~ (Amershum, 116 mCi/mmol) fi~r 8 h at 37°C. The homogenate was then stored at 70°C until use. Isohltion of glycosaminoglycans (GAGs) ['~SSO4]GAGs were prepared from homogenates of metabolically labeled brains and unlabeled GAGs from homogenates of nonlabeled brains, obtained under the same conditions as described above. Lipids were extracted from homogenates according to the technique of Suzuki 4q~ using ice-cold chloroform/methanol (2:1 v/v) and then the reverse ratio of these solvents. The air dried lipid free residual material was rinsed with acetone, ground to a fine powder and stored overnight at 37°C in a dessicator containing P,O~. The powder was dissolved in l0 ml of Tris-HCI, 0.1 M, pH 8.5, CaCI, 4 mM and digested with l0 mg of pronase E (EC 3.4.2.4) (Calbiochem-Boerhinger Corp.) during 72 h at 37°C. The pronase digestion was stopped by boiling at 100°C during 2 rain, The insoluble material was precipitated by centrifugation (10,000× g; 30 rain) and the supernatant was deproteinized with trichloroacetic acid 5% tv/v) at 4°C. DE.4E chromatography The GAG suspension was chromatogranhed on a column of DEAE cellulose equilibrated with Tris-HCI, l0 mM, 0.1 M NaCI, pH 7.5. The column was washed with the e~uilibration buffer, The remaining bound material was eluted with a 0,1-1 M NaCI linear gradient in the same buffer, at a flow rate cf 0.5 ml/min and I ml fractions were collected. In the case of [3"~S]GAG, the radioactivity of the fractions was determined.

Sepharose CL-6B chromatography The fractions containing the radioactively labeled material which eluted between 0.2-0.35 M NaCI from the DEAE cellulose column were pooled and chromatographed on a Sepharose CL-6B column (0.9.',

Embryonic brain-derived heparan sulfate inhibits cellular membrane binding and biological activity of basic fibroblast growth factor.

We have investigated the ability of glycosaminoglycans from embryonic chick brain (15 days old) to interact with basic fibroblast growth factor (bFGF)...
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