Cell surface heparan sulfate proteoglycans Target and partners of the basic ®broblast growth factor in rat Sertoli cells Sylvie Brucato, Jean Bocquet and Corinne Villers Laboratoire de Biochimie, IRBA, Universite  de Caen, France Basic ®broblast growth factor (bFGF) regulates diversi®ed biological functions in rat Sertoli cells. This report demon- strates t hat bFGF inhibits steroidogenesis in developing rat Sertoli cells. Follicle stimulating horm one (FSH)-stimulated estradiol p roduction was reduced by bFGF. M oreover, the amount of cytochrome P450 aromatase, responsible for the irreversible transformation of androgens into estrogens, is decreased by b FGF at the transcriptional l evel. The bFGF inhibitory eect was also observed in the presence of dibu- tyryl-cAMP, ch olera toxin or R O-20-1724, all inducing high levels of cAMP, the second messenger of FSH. Heparan sulfate proteoglycans ( HSPGs) were shown t o be required as cofactors for bFGF signaling. Indeed, sodium chlorate, d escribed to drastically decrease prote oglycan sul- fation, abolishes the bFGF do wnregulation of FSH-stimu- lated estradiol synthesis p reviously observed. Glypican-1, syndecan-1 and -4, potential bFGF coreceptors, are m ainly regulated at the transcriptional level. This report s hows that the b FGF r egulation of their expression speci®cally depends on the nature of HSPG and of the Sertoli cell developmental stage. In conclusion, HSPG are partners and the target of bFGF in rat Sertoli cells. Keywords: bFGF; aromatase;heparan sulfate proteoglycans; RT-PCR; Sertoli cells. The basic ®broblast growth factor (bFGF or FGF-2) belongs t o a large F GF fam ily of 21 structurally related members [1]. This growth factor is produced by many cell types and tissues, i ncluding testis [2]. Its biological activity is pleiotropic [3] as it in¯uences aspects of both cellular growth, differentiation but also angiogenesis, tissue repair and cell migration. In rat testis, bFGF affects, for instance, Leydig and Sertoli cell s teroidogenesis [4,5], Sertoli cell transferrin production [6] and plasminogen activator activity [7] but also c-fos [8] and FGFR-1 [9] mRNA expression. The biological activity of bFGF is mediated by interac- tion with high af®nity cell surface bFGF receptors (FGFR-1 to FGFR-4) [ 10]. In addition, bFGF b inds to h eparan sulfate proteoglycans (HSPG) on the cell surface [11]. Oligosaccharidic sequences of HS chains are de®ned for the bFGF binding and for the recognition of the speci®c bFGF receptor, leading to the formation of a ternary complex comprising HSPG±bFGF±FGFR. These oligosaccharidic motifs are differently sulfated related to the synthesis pathway itself and depending on the cell type. The r esulting structural microheterogeneity modulates bFGF af®nity for its coreceptor and, as a consequence, the growth factor activity. Studies indicated that bFGF binding to HSPG facilitates bFGF receptor binding and activation. bFGF receptor binding to cells that do not express HSPG is signi®cantly reduced when compared to cells expressing HSPG [4,11±15]. Sertoli cells are the principal source of estradiol produc- tion in the immature testis [16,17]. Signi®cant estrogen synthesis is present in Sertoli cells of early postnatal rats, with a sharp reduction during subsequent maturation [18,19]. The p resent work ®rstly aims to evaluate the effect of bFGF on follicle stimuling hormone (FSH)-estradiol syn- thesis and cytochrome P 450 aromatase mRNA expression in 20 days old-rat Sertoli cells. The involvement of t he cAMP pathway w as evaluated using three approaches, all inducing d ifferently high levels of cAMP: (a) dibutyryl cyclic AMP (dbcAMP), a structural analogue of cAMP; (b) cholera t oxin, a protein G s a ctivator; and (c) R O-20±1724, a speci®c phosphodiesterase inhibitor. Then, w e i nvestigated bFGF effect on FSH-estradiol synthesis in t he absence of H SPG in 20-day-old-rat S ertoli cells. These cells were treated with sodium chlorate to completely inhibit sulfatation of proteoglycans and, in consequence, abolish bFGF binding to HSPG. Our previous studies indicated that in immature rat Sertoli cells, cell surface proteoglycans are mainly represented by HSPG [20,21] and among these, at least glypican-1, syn- decan-1 and syndecan-4 mRNAs are e xpressed [22]. M ore- over, syndecan-1 [23], syndecan-4 [24] and glypican-1 [25] are potential coreceptors of bFGF, and are e ssentially regulated at the transcriptional level [26]. Thus, using a semi-quanti- tative RT-PCR, we had demonstrated in immature Sertoli cells that glypican-1 and syndecan-1 mRNA expression was Correspondence to S. Brucato, Laboratoire de Biochimie, IRBA, Universite  de Caen, Esplanade de la Paix, 14032 Caen cedex, France. Fax:+33231954940,Tel.:+33231566576, E-mail: s_brucato@yahoo.fr Abbreviations: bFGF, basic ®broblast growth factor; FSH, follicle stimulating hormone; HSPG, heparan sulfate proteoglycan; PAPS, phosphoadenosine phosphosulfate; FiRE, FGF-inducible response element; FIN-1, FGF-inducible nuclear protein-1; DMEM, Dul- becco's modi®ed Eagle's medium; RhFGF, recombinant human basic FGF; PA PS, phosphoadenosine phosph osulfate; AMV, avian myelo- blastosis virus. (Received 30 May 2001, revised 1 October 2001, accepted 1 4 November 2 001) Eur. J. Biochem. 269, 502±511 (2002) Ó FEBS 2002 speci®cally upregulated by PKC-activatio n i n c ontrast to syndecan-4 transcription [22]. Until now, nothing has been known about the bFGF regulation of glypican-1 and syndecan-1 and -4 mRNA expression in developing Sertoli cells. Our p resent study demonstrates that bFGF in¯uences this expression in 20-day-old-rat Sertoli cells. During testicular development, the physiology of Sertoli cells i s m odi®ed. The cell proliferation decreases and ceases allowing the establishment of the hematotesticular barrier around the 20th day postpartum. In addition, some enzymatic activities are modulated, such as the aromatase activity which decreases upon ontogenesis. T herefore, the study was extended and considered in 10- and 30-day-old- rat Sertoli cells. This report shows that HSPG are necessary for bFGF signal transduction in acting as coreceptors and that HSPG mRNAs expression is modulated by bFGF itself in developing Sertoli cells. MATERIALS AND METHODS Materials Ovine F SH (oFSH -21) was kindly p rovided b y t he National Institute of A rthritis, Metabolic and Digestive Diseases (Pituitary Hormone Distribution program, Bethesda, MD, USA). Dulbecco's modi®ed Eagle's medium (DMEM), Ham's F12 medium, Trypsin (USP Grade), trizol reagent and DNA mass ladder were from Gibco-BRL (Cergy- Pontoise, France). Collagenase-d ispase was from Boehrin- ger-Mannheim (Meylan, France). Ultroser SF (steroid-free serum substitute) was purchased from IBF-Biotechnics (Villeneuve-La-Garenne, France). Bovine pancreas deoxy- ribonuclease (DNase type I), hyaluronidase (type I-S), testosterone, estradiol 17-b,(Bu) 2 cAMP (N6, 2¢-O-dibutyryl- adenosine 3¢:5¢cyclic monophosphate), cholera toxin, Ro 20±1724, sodium chlorate, Hoescht 33258, calf thymus DNA and agarose were purchased from Sigma (Saint- Quentin Fallavier, France). Avian myeloblastosis virus (AMV) reaction buffe r 5 ´, oligo d(T) 15, dNTPs, RNasin, AMV-re verse transcr iptas e, Thermus aquaticus (Taq) DNA polymerase reaction b uffer 1 0 ´, Taq DNA polymerase a nd MgCl 2 were from Promega (Charbonnie Á re-les-bains, France). The oligonucleotide primers were synthesized and puri®ed by Eurobio (Les Ulis, France). Recombinant human basic ®broblast growth factor (RhFGF) were from R & D Systems (Abingdon, UK). 2,4,6,7[ 3 H]-17b estradiol (3.77 TBq ámmol )1 ) was from NEN (les Ulis, France). All reagents were of analytical or molecular biology grade. Cell culture Ten-, 20- and 30-day-old Sprague±Dawley r ats f rom our own colony were killed by cervical dislocation. Sertoli cells were obtained b y sequential enzymatic digestion including trypsin, collagenase and hyaluronidase as described previ- ously [27]. Sertoli cells were seeded at the concentration of 250 000 cellsácm )2 in 24-well dishes or in 75-cm 2 plastic ¯asks and cultured for48 h in Ham's F12/DMEM (1 : 1,v/v) supplemented with 2% Ultroser SF i n order to attach the Sertoli cells in a humidi®ed atmosphere of 5% CO 2 in air at 32 °C. Culture medium was rene wed after 48 h . Three days after plating, residual germinal cells were removed by brief hypotonic treatment using 20 m M Tris/HCl (pH 7.4) [28]. Sertoli cells were then cultured for two days in culture medium devoid of Ultroser before being u sed on d ay 5 a fter plating. For the aromatase assay, Sertoli cells were incubated f or 24 h w ith testosterone (200 ngámL )1 ), oFSH (100 ngámL )1 ) and/or bFGF (5 ngámL )1 ) and/or sodium chlorate (10 m M ). Before RNA extraction, cells were incubated for 24 h either in the absence or in prese nce of FSH, dbcAMP, cholera toxin or bFGF, either in combination with FSH or dbcAMP or cholera toxin and bFGF. Extraction of total RNA Total RNA was extracted from rat Sertoli cells by single step method of Chomczynski & Sacchi [29] using T rizol reagent. T he integrity and quality o f puri®ed RNA were controlled b y 1% agarose gel electrophoresis and measure of the absorbance at 260 and 280 nm. Semi-quantitative RT-PCR Heat denatured total RNA (500 ng; 55±60 °C, 5 min) was added to a reverse transcription r eaction m ixture containing the reaction buffer (50 m M Tris/HCl, pH 8.3, 50 m M KCl; 10 m M MgCl 2 ,0.5m M Spermidine, dithiothreito l 10 m M ), 1 l M oligo d(T) 15 ,500l M dNTPs, 20 UI RNasin, 18 UI AMV-reve rse transcriptase in 20 lL ®nal volume. The reaction was carried out at 37 °C for 60 min and followed by 5 min denaturation at 95 °C. Two microliters of the ®rst strand synthesis product (0.1 lg) wa s used as template to amplify e ach cDNA. PCR was performed with 250 l M dNTPs, Taq DNA polymerase reaction buffer (50 m M KCl, 10 m M Tris/HCl, pH 9; 0 .1% Triton X-100), 2.5 UI Taq DNA polymerase, MgCl 2 1.5 m M ,10pmolofeachprimer(Table1)ina20-lL reaction volume. The PCR was started at 94 °C1minandfollowedbyup to 27 cycles of am pli®cation for t he three proteoglycans and 20 cycles for the internal control, b-actin as described previously [23], which consisted of a denaturating step (at 94 °C f or 1 min), an annealing s tep (at 55 °C f or 1 min) a nd an extension step (at 72 °C for 2 min) then a ®nal elongation step (at 72 °C for 10 min) in ROBOCYCLERÒ Gradient 40 (Stratagene). The cytochrome P450 aromatase c DNA was ampli®ed at 94 °C for 1 min for 3 0 cycles a s described previously [30], which consisted of a denaturating step (at 94 °Cfor1min), an annealing step (at 6 0 °C for 30 s) and an extension step (at 72 °C for 1 min) then a ® nal elongation step (at 72 °C for 10 min) in ROBOCYCLERÒ Gradient 40 (St ratagene). To check for contaminating genomic DNA, a RT-PCR was performed on RNA without AM V reverse tran scriptase (data not shown). In a ll negative P CR control r eactions, cDNA templates were replaced with sterile wate r to check the absence of contaminants. Aliquots ( 10 lL) of the P CR reaction w ere size- separated on a 4% agarose gel equilibrated in Tris/ acetate/EDTA (40 m M Tris/acetate, 1 m M EDTA). Gels were stained w ith e thidium b romide (1 lgámL )1 ), photo- graphed using Polaroid ®lm under UV light and ana- lysed using a AGFA SnapScan 1200 P Scanner Ò , Adobe Ó FEBS 2002 HSPG, target and partners of bFGF (Eur. J. Biochem. 269) 503 PHOTOSHOP Ò software and the NIH IMAGE computer program (http://rsb.info.nih.gov/nih-image). Radio immuno assay of estradiol 17-b Culture medium was extracted with 5 vol. of diethylether and estradiol was quanti®ed by radioimmunoassay using a speci®c antibody purchased from Biosys (Compie Á gne, France). The only signi®cant cross reactions were for 2-methoxy-estradiol ( 5%), e stra diol 17 a (0.28%), estrone and estriol (0.45%). The sensitivity of the assay w as 6 pg per tube. Intra- and interassay coef®cients of variation were less than 10%. The analysis o f the radioimmunoassay data was performed using the SECURIA program from the Packard Instrument Company (Meriden, CT, USA). DNA quanti®cation The DNA content of the cell layer at the end of incubation was quanti®ed by the method of West et al .[31].After solubilization of the cell layer in 1 M NaOH and s ubsequent neutralization by 1 M KH 2 PO 4 , DNA was quanti®ed in a Kontron spectro¯uorimeter u sing Hoescht 33258 as ¯uo - rescent probe and calf thymus as standard. Statistical analysis All experimental datawere presented as the meanof duplicate (estradiol) determinations of three wells in, at l east, three different cultures within e ach treatment grou p. Results were normalized in pg (estradiol) per 10 6 cells. Statistical signi®- cance between groups was determinated by Student's paired t-test. Differe nces were c onsidered signi®cant at p < 0.05. RESULTS Cell surface HSPG are bFGF partners. bFGF inhibits the FSH-stimulated estradiol synthesis in Sertoli cells Sertoli cells from 20-days-old-rats were i ncubated for 24 h with FSH (100 ngámL )1 ) and increasing concentrations of bFGF (0.1±10 ngámL )1 ). FSH-stimulated estradiol synthe - sis was inhibited and appeared to be dose-dependent (Fig. 1 ). The maximal bFGF effect ()49%) on FSH- induced estradiol production was r eached for 5 ngámL )1 .In contrast, estradiol synthesis was not regulated b y bFGF in the absence of FSH (data not shown). Direct implic ation o f cAMP increase, the second mes- senger of FSH, in the bFGF regulation was evaluated by addition to the culture medium of either 1 m M dbcAMP or 10 lgámL )1 cholera toxin. Their addition increased e stradiol production by a factor 9 and 7 , respectively (Table 2) as FSH did (factor 9 ) (Fig. 1). In the presence of dbcAMP or cholera toxin, bFGF addition induces a similar inhibition ()49% and )40%, r espectively) on estradiol synthesis as the one described in Fig. 1 (Table 2). The c AMP level elevation was also aprehended in the presence of 100 ngámL )1 FSH and 250 l M RO 20±1724, a speci®c inhibitor o f cAMP-speci®c phosphodiesterase [32]. In these conditions, a signi®cant increase (about +65%) of estradiol production was obtained (Fig. 2) as already described. We observed that concomittant treatment with 5ngámL )1 bFGF also induced a decrease (about )30%) of FSH-stimulated estradiol synthesis. Nevertheless, this decrease was lesser by comparison to the one observed in the presence of FSH and bFGF ()49%). This result suggested that bFGF action could induce, in part, a Table 1 . Primers for PCR ampli®cation. Target cDNA Localization and sequence of primer Size of PCR products (bp) Syndecan-1 5¢-370 AGGTGCTTTGCCAGATATGACT-3¢ 432 5¢-802 CTCTTTGATGACAGAAGTGCCT-3¢ Syndecan-4 5¢-85 GAGTCGATTCGAGAGACTGA-3¢ 365 5¢-450 AAAAATGTTGCTGCCCTG-3¢ Glypican-1 5¢-566 GAATGACTCGGAGCGTACACTG-3¢ 488 5¢-1054 CCTTTGAGCACATTTCGGCAA-3¢ P450 aromatase 5¢-1555GCTTCTCATCGCAGAGTATCCGG-3¢ 289 5¢-1821CAAGGGTAAATTCATTGGGCTTGG-3¢ b-actin 5¢-2350 ACAGACTACCTCATGAAGAT-3¢ 665 5¢-3222 AGCCATGCCAAATGTCTCAT-3¢ Fig. 1. Dose-related eect of bFGF on FSH-stimulated estradiol syn- thesis in immature cultured rat Sertoli cells. Sertoli cells were incubated for 24 h with testosterone substrat e (200 ngáml )1 ) in the presence or not (c ontrol) of FSH (100 ngáml )1 ) and of bFGF increasing conc en- trations. Determination of estradiol production was pe rformed by radioimmunoassay. Values are expressed in p g per 10 6 cells and are representative o f three experiments (mean  SEM). **, Signi®cantly dierent at P< 0.01; ***, s igni®cantly dierent at P <0.001from FSH values. NS, not signi®cant. 504 S. Brucato et al. (Eur. J. Biochem. 269) Ó FEBS 2002 decrease of FSH-stimulated estradiol s ynthesis by stimulat- ing cAMP-speci®c phosphodiesterase activity. bFGF inhibits the FSH-stimulated cytochrome P450 aromatase mRNA expression The relative expression of cytochrome P450 aromatase mRNA was evaluated using semi-quantitative RT-PCR. In the presence of 100 ngámL )1 FSH, cytochrome P450 aromatase mRNA expression was highly increased as described previously [30]. Sertoli cells from 20-day-old-rats were then incubated for 24 h w ith 100 ngámL )1 FSH and 5ngámL )1 bFGF. In these conditions, cytochrome P450 aromatase mRNA e xpression was inhibited ()41%) by comparison to FSH taken as control (Fig. 3). A similar bFGF inhibitory effect on P450 aromatase mRNA e xpres- sion was observed in the presence of dbcAMP ()41%) or cholera toxin ()45%) (data not shown). bFGF effect on FSH stimulated steroidogenesis requires the presence of HSPG We examined in what extend sodium chlorate treatment could m odify inhibitory effect of exogenous bFGF on FSH- stimulated estradiol synthesis. Indeed, Sertoli cells are bFGF pr oducing c ells [2,33] and are the t arget of this growth factor as bFGF inhibits FSH-induced estradiol synthesis ([5], and our results). HSPG and especially glypicans and synde cans have been described as coreceptors for this growth factor via a highly sulfated sequence of their heparan sulfate chains [34]. Sodium chlorate is an inhibitor of ATP sulfurylase and hence of the production of phosphoadenosine phospho- sulfate (PAPS), the active s ulfate donor for s ulfotrans- ferases. It has been shown to abolish sulfation on proteins and carbohydrate residues in intact cells without inhibiting cell growth or protein synthesis [35±37], and proteoglycan sulfation in cultured Sertoli cells from 20-day-old rats [38]. When Sertoli cells were incubated with 1 0 m M sodium chlorate for 24 h, an increase of FSH-stimulated e stradiol production (+42.5%) w as observed a s d escribed previously [38]. A ddition of 10 m M NaCl, u sed as negative control, did not induce any modi®cation of FSH-stimulated estradiol synthesis (data not shown). H owever, c oncomitant treat- ment of bFGF with 10 m M sodium chlorate totally abolished the in hibitory b FGF e ffect previously observed on Sertoli cell estradiol synthesis (Fig. 4). Similar results were obtained when Sertoli cells were incubated with 1 m M dbcAMP instead of F SH (data not shown). Thus, abolition b y sodium chlorate of bFGF effe ct on FSH-stimulated steroidogenesis could implicate HSPG in t he bFGF signaling. bFGF effect on cell attachment Addition of 5 ngámL )1 bFGF did not promote any signi®cant difference in cell attachment to substratum (data not shown) as the DNA content o f the cell layer at the end of the 24 h incubation period was i dentical in untreated and bFGF-treated Sertoli cell cultures (2525  257 and 2538  246 ng per well in three different cell cultures). Cell surface HSPG are bFGF partners during Sertoli cell postnatal development Developing Sertoli cells undergo structural, b iochemical and functional modi®cations as previously mentioned. Thus, the relationship between bFGF and H SPG was evaluated in Sertoli cells from 10 to 30-days-old-rats. bFGF effect on Sertoli cell estradiol synthesis When Sertoli cells were incubated for 24 h with 100 ng ámL )1 FSH, estradiol production decreased with developing Sertoli cells (Table 3). When Sertoli cells from 10-day-old-rats were incubated for 24 h with 100 ng ámL )1 FSH and 5 ngámL )1 bFGF, FSH-stimulated estradiol production decreased ()40%). This inhibition was less important than in Sert oli cells from 20- and 30-day-old rats ()49 and )53%, respectively) (Table 3). Thus, bFGF inhibitory e ffect was m ore pro- nounced on FSH-stimulated estradiol production with Table 2. bFGF eect on cAMP-stimulated estradiol synthesis in immature cultured ra t Sertoli cells. Sertoli cells were incubat ed for 24 h with testosterone substrate (200 ngámL )1 ) (control) in the presence of 1m M dbcAMP or 10 lgámL )1 cholera toxin and/or 5 ngámL )1 of bFGF. Estradiol production was determined by radioimmunoassay. Values are e xpressed in pg per 1 0 6 cells an d a re rep r esentative of three experiments (mean  SEM). Estradiol (pg per 10 6 cells) Control 53  4 dbcAMP (1 m M ) 480  2 dbcAMP (1 m M ) + bFGF (5 ng mL )1 ) 244  16 Cholera toxin (10 lgá mL )1 ) 379  29 Cholera toxin (10 lgá mL )1 ) + bFGF (5 ngámL )1 ) 227  18 Fig. 2. Inhibitory eect of bFGF o n FSH-stimulated estradiol synthesis by Sertoli cells cultured in the presence of Ro-20-1724, a cAMP phos- phodiesterase inhibitor. Sertoli cells were incubated for 24 h with testosterone substrate (200 ngáml )1 ), FSH ( 100 ngáml )1 )and/orRo-20- 1724 (250 lM) and /or bFGF (5 ngáml )1 ). Estradiol production was determined by radioimmunoassay. Valu es are expressed in pg p er 10 6 cells and are representative of three e xperiments (mean  SEM). *, signi®cantly diere nt at p < 0 .05 from FSH or F SH + Ro-20- 1724 values. Ó FEBS 2002 HSPG, target and partners of bFGF (Eur. J. Biochem. 269) 505 Sertoli cells aging. Moreover, cytochrome P450 aromatase mRNA expression was regulated similarly by bFGF upon development (data not shown). We suggest that bFGF could participate in the Sertoli cell steroidogenesis decrease by inhibiting cytochrome P450 aromatase mRNA expres- sion and FSH-stimulated estradiol production. bFGF effect on steroidogenesis in the absence of cell surface HSPG When Sertoli cells from 10- to 30-day-old-rats were incubated with 100 ngámL )1 FSH, 1 0 m M sodium chlorate and 5 ngámL )1 bFGF for 24 h , bFGF inhibitory e ffect on Fig. 3. Dose-related eect o f bFGF on F SH-stimulated P450 aromatase mRNA in immature cultured rat S ertoli cells. Sertoli cells were incubated for 24 h in the absence of 100 ngáml )1 FSH ( lane 1) or in the presence o f 100 ngáml )1 FSH and increasing concentrations of bFGF (lanes 2 to 6). Total RNA was extracted as described in Materials a nd m ethods. Then, 500 ng RNA was reverse-transcribed and am pli®ed by relative quantitative RT-PCR as previously described. (A) Agarose gel of one representative experiment. (B1) The densitometry data are representative of three experiments (mean  SEM). Aromatase mRNA level under treatment is expressed versus control which is arbitrarily set t o 100%. (B2) The densitometry data are representative of three experiments (mean  SEM ). Aromatase mRNA level under treatment is expressed versus FSH which is arbitrarily set to 100%. 506 S. Brucato et al. (Eur. J. Biochem. 269) Ó FEBS 2002 FSH-stimulated estradiol synthesis was not observed (Table 3). In conclusion, bFGF requires c ell surface HSPG for t he inhibition of steroidogenesis in developing Sertoli cells. Cell surface HSPG are bFGF targets during Sertoli cell postnatal development As previously mentioned, glypican-1, syndecan-1 and syndecan-4 are coreceptors for bFGF in some cellular models. As bFGF requires cell surface HSPG to regulate FSH-stimulated estradiol p roduction, we evaluated if bFGF itself could in¯uence s yndecan-1, syndecan-4 and glypican-1 mRNAs expression. bFGF effect on glypican-1, syndecan-1 and syndecan-4 mRNAs expression TherelativemRNAexpressionoftheseHSPGwas evaluated using semi-quantitative RT-PCR a s d escribed previously [22]. Figure 5 indicated that, when Sertoli cells from 10-, 20- and 30-day-old-rats were incubated for 24 h without any treatment, glypican-1 mRNA expression was signi®cantly increased between 10- and 20-days old, t hen unchanged between 20- and 30-days old. Syndecan-1 mRNA expression was the same whatever rat age. Syndecan-4 mRNA expression increased highly between 10- a nd 20-days old, then decreased between 20- and 30-days-old but however, w as higher than at 10 days old ( Fig. 5). When Sertoli cells from 10-day-old-rats were incubated for 24 h in the presence of 10 ngámL )1 bFGF, b FGF inhibited glypican-1 mRNA expression ()35%) but had no e ffect on syndecan-1 mRNA expression. On the other hand, bFGF stimulated syndecan-4 mRNA expression (+ 41%) (Fig. 6). When Sertoli cells from 20-day-old rats were incubated for 24 h in the presence of 10 ngámL )1 bFGF, glypican-1 mRNA expression was inhibited ()37%) similarly to 10-day-old cells whereas syndecan-1 and syndecan-4 mRNAs e xpression was not modi®ed c ompared to t he control (Fig. 6). In Sertoli cells from 30-day-old rats, bFGF had no inhibitory effect on glypican-1 mRNA expression, but increased syndecan-1 and syndecan-4 mRNA expression (+36% and +42%, respectively) (Fig. 6). DISCUSSION This report shows for the ®rst time in developing rat Sertoli cells that HSPGs are partners for b FGF signal t ransduction as coreceptors, a nd that HSPGs are a target of this growth factor as their mRNA expression is modulated by bFGF itself. Under our cell culture conditions, 20-day-old-rat Sertoli cells did not proliferate. Thus, bFGF effect was evaluated by estradiol synthesis instead of cell proliferation test. We demonstrated that bFGF regulates steroidogenesis upon cell development. Thus, bFGF d ecreased c ytochrome P 450 aromatase mRNA e xpression but also inhibited the FSH- stimulated estradiol synthesis. Therefore, the mechanism by which bFGF inhibits Sertoli cell steroidogenesis is still unknown in developing rat Sertoli cells. However, it seems that bFGF mainly regulates steroidogenesis at the tran- scriptional l evel and could also, in part, stimulate phospho- diesterase activity as TGF-b does [ 39]. Thus, bFGF, among other testicular agents, could participate in the decrease of this Sertoli cell FSH-stimulated estradiol production during t estis development [40,41]. In this way, the i nhibitory effect of bFGF on estradiol production may represent signals destined to shut down aromatase activity because recent observations made by Sharpe et al. [42] have led to the conclusion that prolonged exposure o f Sertoli cells to estrogens impairs or delays their functional maturation. Fig. 4. Eect of bFGF in the presence of sodium chlorate on FSH- stimulated estradiol synthesis in immature rat Sertoli cells. Sertoli cells were incubate d with t estoste rone su bstrate ( 200 ngáml )1 )(control)in the p resence of FSH (100 ngámL )1 )and/orbFGF(5ngámL )1 )and/or sodium chlorate (10 m M ) during 2 4 h . Estradiol pro duction was determined by radioimmunoassay. Valu es are expressed in pg per 10 6 cells and are represen tative of t hree experiments (mean  SEM). **, Signi®cantly di erent at P < 0.01 f ro m FSH values. NS, n ot signi®cant. Table 3. bFGF eect on FSH-stimulated estradiol synthesis in the presence or not of c ell surface HSPG during S ertoli cell development. Sertoli c ells were incubated for 24 h with testosterone substrate (200 ngámL )1 ) in the p resence or not (control) of F SH (100 ngámL )1 )and/orbFGF(5ngámL )1 ) and/or sodium chlorate ( 10 m M ). Estradiol production was determined by radioimmunoassay. Values are expressed i n pg per 10 6 cells and are representative of three experiments (mean  S EM) for each studied age. Sertoli cells (days old) Estradiol synthesis (pg per 10 6 cells) Control FSH FSH + bFGF FSH + chlorate FSH + chlorate + bFGF 10 760  64 1220  27 830  50 1490  70 1380  100 20 53  4 458  28 234  3 653  12 578  69 30 25  0.6 111  9 52  5 170  22 147  15 Ó FEBS 2002 HSPG, target and partners of bFGF (Eur. J. Biochem. 269) 507 The bFGF binding requires speci®c motifs on highly sulfated HS chains. Sodium chlorate, in inducing structural alteration of HS chains, prevents the bFGF binding. Our results indicate that the presence of sodium chlorate abolishes the bFGF biological effects in d eveloping Sertoli cells. Thus, inhibitory effect of bFGF on FSH-stimulated estradiol production requires t he presence of cell surface HSPG with correctly sulfated HS chains in addition to bFGF receptor as shown in previous studies [4,11±15]. Among these HSPG, glypican-1, syndecan-1 and syndecan-4 are potential coreceptors o f bFGF. We indicated that the HSPG mRNA pattern is not similar in Sertoli cells from 10, 20 a nd 30 days old. In addition, glypican-1 mRNA expression was inhib ited ()35% and )37%, respectively) in Sertoli cells f rom 10- and 20- day-old rats as d escribed in oligodendrocytes [43] or in lung ®broblasts [44]. I n contrast, this inhibition was abolished in 30-day-old rat Sertoli cells. Syndecan-1 mRNA expression was not modi®ed by bFGF in Sertoli cells from 10 and 20-day-old rats whereas it was stimulated in Sertoli cells from 30-day-old rats. If bFGF stimulated syndecan-4 mRNA expression in Sertoli cells from 10 and 30-day-old rats, no effect was observed in Sertoli cells from 20-day-o ld rats. Thus, bFGF effect seems to be HSPG-, developmental stage- and cell type-speci®c. Indeed, bFGF does not regulate syndecan-1 mRNA expression in MCA3D keratinocytes [45,46] or in endothelial cells [47]. However, this growth f actor increases syndecan-1 and syndecan-4 m RNAs expression in ®broblasts [48] and in vascular smooth muscle cells [49], respectively. At the transcriptional level, t he 5 ¢ ¯anking r egion o f syndecan-1 contains an FGF-inducible response element (FiRE) [50]. In this study, bFGF increases syndecan-1 mRNA expression in Sertoli cells from 30-day-old r ats. This observation suggests that these cells might express all transcription facto rs comp onents o f F iRE, namely USF, the uncharacterized p46 nuclear proteins, A P-1 (Jun/Fos) complexes and a putatively novel FGF-inducible nuclear protein-1 (FIN-1) [50]. T hese transcriptional elements are differentially regulated d epending on cell type and activating growth factor [50]. In NIH 3T3 ®broblasts, FiRE w as shown to be selectively induced by bFGF whereas in keratinocytes, FiRE was not induced by this growth factor. Whether FiRE is really expresse d in Sertoli cells or not, some post-translational modi®cations, phosphorylation or dephosphorylation of FiRE components could contribute to the speci®city [51±54]. Inhibitory transcription factors that bind to AP-1 or FIN-1 o r that inactive b inding or transactivation capacity of, for example, FIN-1 and USF-1, could explain activation or inhibition of FiRE. From our results, it seems that bFGF r egulates differently HSPG expression upon Sertoli cell maturation suggesting a func- tional selectivity. Assuming that levels of glypican-1 and syndecan protein synthesis c orrelate well with mRNA s l evels, it is likely that, in the p resence of b FGF, the plasma membrane will be enriched with syndecans and a decrease of glypican-1. Further experiments will be needed to understand the biological signi®cance of t he different regulation o f their expression. As a ®rst element step t owards understanding, a recent demonstration indicated that the synthesis of cell surface HSPG and FSH-stimulated estradiol synthesis are inversely correlated. This suggests a potential role for these Fig. 5. Evolution o f glypican-1, syndecan-1 and syndecan-4 mRNAs expression during Ser toli cells development. Sertoli cells from 10-, 20- and 30-day-old rats w ere incubated for 24 h without treatment. T otal RNA was extracted as described in M aterials and methods. Then RNA (500 ng) was reverse transcribed a nd ampli®ed by relative q uantitative RT-PCR as described previously [ 22]. Glyp-1, glypican-1; Synd-1, syndecan-1; Synd-4, syndecan-4. (A) Agarose gels of one representative experimen t. (B) Densitometry data are representative of ®ve experiments (mean  SE) for each age. 508 S. Brucato et al. (Eur. J. Biochem. 269) Ó FEBS 2002 HSPG in the decrease of estradiol production [55]. 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