Báo cáo Y học: Regulation of glypican-1, syndecan-1 and syndecan-4 mRNAs expression by follicle-stimulating hormone, cAMP increase and calcium influx during rat Sertoli cell development pptx

Regulation of glypican-1, syndecan-1 and syndecan-4 mRNAs expression by follicle-stimulating hormone, cAMP increase and calcium influx during rat Sertoli cell development Sylvie Brucato, Jean Bocquet and Corinne Villers Laboratoire de Biochimie IRBA, UPRES, Universite ´ de Caen, France In seminiferous tubules, Sertoli cells provide structural and nutritional support for the developing germinal cells. Cell- to-cell signaling and cell adhesion require proteoglycans expressed at the cell membrane. A preliminary biochemical and structural approach indicated that cell surface proteo- glycans are mostly heparan sulfate proteoglycans (HSPG). Glypican-1, syndecans-1 and -4 were identified using a molecular approach. Their differential regulation was dem- onstrated in immature rat Sertoli cells. Follicle-stimulating hormone (FSH) is the main regulator of Sertoli cell function. Signal transduction triggered by FSH involves both an increased intracellular cAMP synthesis and a calcium influx. This study demonstrates that FSH, through its second messengers (increase in intracellular cAMP and intracellular calcium), downregulated the glypican-1 mRNA expression in Sertoli cells from 20-day-old rats. On the other hand, syndecan-1 mRNA expression is not modulated by FSH as it would result from the antagonistic effects of increased intracellular cAMP and intracellular calcium levels. Finally, syndecan-4 mRNA expression is not regulated by this pathway. The present study was extended during Sertoli cell devel- opment. Indeed, Sertoli cells undergo extensive changes during the postnatal period both in structure and function. These important transformations are critical for the esta- blishment of spermatogenesis and development of the adult pattern of testicular function. Our data indicated that the regulation of HSPG mRNA expression is HSPG-specific and depends on the Sertoli cell developmental stage. Keywords: 1 FSH; calcium; heparan sulfate proteoglycan; Sertoli cell development. Syndecans and glypicans are cell surface receptors bearing heparan sulfate (HS) chains and comprising four (syndecan-1 to -4) [1,2] and six members (glypican-1 to -6) [3], respectively. Syndecans are characterized by a specific extracellular domain displaying low sequence homology, and highly conserved transmembrane and cytoplasmic domains. How- ever, the cytoplasmic domain of all four syndecans contains a central region unique to each syndecan, which would confer specific biological activity [4,5]. Glypicans are attached to the plasma membrane via glycosylphophatidylinositol (GPI) anchors [1,2]. Although the primary structure of the glypi- cans is only marginally conserved (about 30% identity), there is a strict conservation of 14 cysteine residues within the core protein leading to compact conformation but also of HS-attachment consensus sequences at the C-termini of proteins. Syndecans and glypicans are individually expressed in distinct cell-, tissue-, and development-specific patterns [6–10]. Modifications in glypican and syndecan expression may be induced by activation of signaling processes. It was shown that glypican-1 is downregulated by the presence of both bFGF and TGF-b1 in fibroblasts [11] and by bFGF in mature oligodendrocytes [12], but the mechanisms that account for the regulated expression of the glypican are almost completely unknown. In contrast, syndecan expres- sion is modulated by growth factors and cytokine [13,14]. In most cells, levels of syndecan synthesis correlate well with syndecan mRNA levels, suggesting that the regulation is mainly transcriptional [8]. Syndecans and glypicans bind a variety of extracellular matrix molecules and growth factors in a heparan sulfate dependent manner [15]. Consequently, they take part in the regulation of various biological events. In addition, synde- cans associate with the actin cytoskeleton through a mechanism dependent on their cytoplasmic domains [16]. Syndecan-4, which presents the widest expression pattern, is localized to the focal adhesions of a range of cells in a protein kinase C (PKC)-dependent manner and may function as a coreceptor with integrins [17]. The variable cytoplasmic region unique to syndecan-4 interacts with the catalytic domain of PKCa and stimulates its activity [18]. These observations strongly suggest the participation of syndecans in signal transduction mechanisms while that of glypicans is still poorly understood. In the mammalian testis, Sertoli cells are epithelia somatic cells associated, by a basement membrane, to peritubular cells surrounding seminiferous tubules. They play a crucial Correspondence to S. Brucato, Laboratoire de Biochimie IRBA, UPRES A 2608, Universite ´ de Caen, Esplanade de la Paix, F-14032 Caen cedex, France. Fax: + 33 02 31 95 49 40, Tel.: + 33 02 31 56 65 76, E-mail: s_brucato@yahoo.fr Abbreviations: HS, heparan sulfate; HSPG, heparan sulfate surface proteoglycan; FSH, follicle-stimulating hormone; GPI, glycosylpho- phatidylinositol; BHT, hematotesticular barrier; DMEM, Dulbecco’s modified Eagle’s medium; FiRE, bFGF-inducible response element; VSM, vascular smooth muscle; CREB, cAMP response element binding protein; CREM, cAMP response element modulator. (Received 18 February 2002, revised 8 May 2002, accepted 29 May 2002) Eur. J. Biochem. 269, 3461–3469 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03027.x role in the spermatogenesis process in providing structural support and specialized microenvironment essential for germ cell differenciation [19]. The physical and functional interactions between these different cell types require specific molecules. Among these molecules, heparan sulfate proteo- glycans (HSPG) may represent agents of great importance due to their cell localization and properties. Our previous studies have shown that cell surface proteoglycans are mainly represented by HSPG in immature rat Sertoli cells [20,21]. These HSPG are involved in more differentiated function as Phamanthu et al. [22,23] have shown that alteration of proteoglycan synthesis and sulfation enhanced follicle-stimulating hormone (FSH)-stimulated estradiol synthesis by regulating phosphodiesterase activity. Among these Sertoli cell HSPGs, at least glypican-1, syndecan-1 and syndecan-4 mRNAs are expressed and regulated by PKC activation [24] and bFGF [25]. However, little is known about factors that might regulate their expression in these cells. FSH is the main regulator of Sertoli cells functions. Although no FSH effect on the whole proteoglycans synthesis has been described [26], FSH regulation on specific HSPG mRNAs expression can not be excluded. The binding of FSH onto specific receptors on Sertoli cells leads to an intracellular cyclic adenosine monophosphate (cAMP) increase [27,28]. In addition, the entry of extracellular calcium is involved in the signal transduction triggered by FSH in Sertoli cells [29–31]. It has been clearly demonstra- ted that calcium influx occurs through voltage-independent and -dependent calcium channels [30,31]. The latter have beenidentifiedasbothLandNtype[30,32,33]. During testicular development, the physiology of Sertoli cells is modified. The cell proliferation decreases and ceases allowing the establishment of the hematotesticular barrier (BHT) at around day 20 postpartum. Sertoli cells progres- sively lose FSH responsiveness. In addition, some enzymatic activities are modulated as the aromatase activity decrease or the cAMP phosphodiesterase increase upon ontogenesis [19]. The present work aims to investigate whether FSH, intracellular cAMP level increase and alteration of trans- membrane calcium influx induce changes on glypican-1, syndecans-1 and -4 mRNA expression in developing rat Sertoli cells. MATERIALS AND METHODS Reagents All reagents were of analytical or molecular biology grade. Dulbecco’s modified 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-dispase was from Boehringer–Mann- heim (Meylan, France). Ultroser SF (steroid-free serum substitute) was purchased from IBF-Biotechnics (Ville- neuve-La-Garenne, France). Bovine pancreas deoxyribo- nuclease (DNase type I), hyaluronidase (type I-S), agarose, dbcAMP, cholera toxin, Ro-20-1724, H8 {N-[2-(methyl- amino)ethyl]-5-isoquinoline-sulfonamide} and verapamil were purchased from Sigma (Saint-Quentin Fallavier, France). oFSH is a generous gift of National institute of Arthritis Metabolic and Digestive Diseases. Avian Myelo- blastosis Virus (AMV) reaction Buffer 5·, oligo d(T) 15, dNTPs, RNasin, AMV-reverse transcriptase, Thermus aquaticus (Taq) DNA polymerase reaction buffer 10·, Taq DNA polymerase and MgCl 2 were from Promega (Char- bonnie ` re-les-bains, France). The oligonucleotide primers were synthesized and purified by Eurobio (Les Ulis, France). Cell culture Sprague–Dawley rats (10-, 20- and 30-days-old), obtained from our own colony, were killed by cervical dislocation. Sertoli cells were obtained by sequential enzymatic digestion including trypsin, collagenase and hyaluronidase, as des- cribed previously [34]. Sertoli cells were seeded at the concentration of 250 000 cellsÆcm )2 in 75 cm 2 plastic flasks and cultured 48 h in Ham’s F12/DMEM (1 : 1, v/v) supplemented with 2% Ultroser SF in order to attach the Sertoli cells in a humidified atmosphere of 5% CO 2 in air at 32 °C. Culture medium was renewed 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) [35]. The culture flasks were washed with fresh medium without Ultroser SF. Monolayer Sertoli cells were used on day 5 after plating. They were incubated for 24 h either in absence or in presence of various treatments before RNA extraction. Extraction of total RNA Total RNA was extracted from rat Sertoli cells by single step method of Chomczynski & Sacchi [36] using trizol reagent. The integrity and quality of purified RNA were con- trolled by 1% agarose gel electrophoresis and measure of the absorbance at 260 and 280 nm. Semi-quantitative RT-PCR Denatured total RNA (500 ng, 55–60 °C, 5 min) was added to a reverse transcription reaction mixture 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, dithiothreitol 10 m M ), 1 l M oligo d(T) 15 , 500 l M dNTPs, 20 U RNasin, 18 U AMV-reverse transcriptase in 20-lL final volume. The reaction was carried out at 37 °C for 60 min, followed by 5 min denaturation at 95 °C. Two microliters of the first strand synthesis product (0.1 lg) was used as template to amplify each 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 ,10pmolofeachprimerina20-lL reaction volume. The sequences of 5¢ and 3¢ primers were 5¢-AGGT GCTTTGCCAGATATGACT-3¢ and 5¢-CTCTTTGATG ACAGAAGTGCCT-3¢ for syndecan-1; 5¢-GAGTCGAT TCGAGAGACTGA-3¢ and 5¢-AAAAATGTTGCTGCC CTG-3¢ for syndecan-4; 5¢-GAATGACTCGGAGCGTAC ACTG-3¢ and 5¢-CCTTTGAGCACATTTCGGCAA-3¢ for glypican-1; 5¢-ACAGACTACCTCATGAAGAT-3¢ and 5¢-AGCCATGCCAAATGTCTCAT-3¢ for b-actin. The PCR was started at 94 °Cfor1minandfollowedby up to 27 cycles of amplification for the three proteoglycans and 20 cycles for the internal control, b-actin as described 3462 S. Brucato et al. (Eur. J. Biochem. 269) Ó FEBS 2002 previously [24] which consisted of a denaturating step (at 94 °C for 1 min), an annealing step (at 55 °Cfor1min) andanextensionstep(at72°C for 2 min) then a final elongation step (at 72 °C for 10 min) in RobocyclerÒ Gradient 40 (Stratagene). Optimum RT-PCR conditions were established in order to further determine possible regulations of the HSPG mRNA expression (constant input cDNA, determination of optimal cycle number) [24]. An RT-PCR was performed without AMV reverse transcriptase in order to check for contamin- ating genomic DNA (data not shown). In all negative PCR control reactions, cDNA templates were replaced with sterile water to check the absence of contaminants. Ten-microliter aliquots of the PCR reaction were size- separated on a 4% agarose gel equilibrated in Tris/acetate/ EDTA (0.04 M Tris, acetate, 0.001 M EDTA). Gels were stained with ethidium bromide (1 lgÆmL )1 ), photographed using Polaroid film under UV light and analysed using a AGFA SnapScan 1200 2 P ScannerÒ, ADOBE PHOTOSHOP Ò software and the NIH IMAGE computer program (http:// rsb.info.nih.gov/nih-image). DNA quantification The DNA content of the cell layer at the end of incubation was quantified by the method of West et al. (1985) [37]. After solubilization in 1 M NaOH of the cell layer and subsequent neutralization by 1 M KH 2 PO 4 , DNA was quantified in a Kontron spectrofluorimeter using Hoescht 33258 as fluorescent probe and calf thymus as standard. RESULTS FSH inhibits glypican-1 mRNA expression in Sertoli cells from 20-day-old rats Sertoli cells from 20-day-old rats were incubated for 24 h with increasing concentrations of FSH (10–200 ngÆmL )1 ). FSH did not modify significantly syndecan-1 and -4 mRNAs expression (Fig. 1). In contrast, glypican-1 mRNA expression decreased in a dose-dependent manner. The optimal effect was obtained from 100 ngÆmL )1 of FSH corresponding to a 45% decrease in the glypican-1 mRNA expression (Fig. 1). Glypican-1 and syndecans-1 and -4 mRNA expression are regulated by the increase of intracellular cyclic AMP and calcium level in Sertoli cells from 20-day-old rats FSH stimulates at least two signaling pathways in the Sertoli cells. This hormone induces the increase of intracel- lular cyclic AMP and calcium levels. Effect of intracellular cAMP level increase The involvement of the cAMP pathway was evaluated using three approaches, all inducing high levels of cAMP: (a) dibutyryl cyclic AMP (dbcAMP), a structural analogue of cAMP; (b) cholera toxin, a protein G s activator [38]; and (c) RO-20 1724, a specific cAMP phosphodiesterase inhib- itor [39]. Twenty-day-old rat Sertoli cells were incubated for 24 h with increasing concentrations of dbcAMP (0–2 m M )(data not shown). The mRNA glypican-1 expression inhibition was optimal ()56%) for 1 m M of dbcAMP and maintained for high concentrations of dbcAMP. The syndecan-1 mRNA expression was increased by 1 m M of dbcAMP (+50%) whereas the syndecan-4 mRNA expression was not modulated in Sertoli cells from 20-day-old rats (Fig. 2). In a second experiment, the dbcAMP effect was con- firmed by using 10 lgÆmL )1 of cholera toxin during 24 h in 20-day-old rat Sertoli cells. This agent induced the same effects as the dbcAMP ones. Indeed, cholera toxin inhibited glypican-1 mRNA expression ()49%), and increased the one of syndecan-1 (+50%) but had no significant effect on syndecan-4 mRNA expression (Fig. 3). Finally, RO-20 1724, a specific cAMP phosphodiesterases inhibitor used at 250 l M , led to similar results as the ones described in Figs 2 and 3. The glypican-1 and syndecan-1 mRNAs expression were significantly decreased ()39%) and increased (+36%), respectively, whereas syndecan-4 mRNA was not significantly affected by the treatment (Fig. 4). The optimal doses of chemical compounds used in this study are doses that induce various effects in Sertoli cell culture as proteoglycan synthesis [53] or estradiol production [25]. As a first conclusion, in immature Sertoli cells, the increase of intracellular cAMP level (a) regulates the glypican-1 and syndecan-4 mRNA expression as FSH does; and (b) modulates syndecan-1 mRNA expression in contrast to FSH effect. Nevertheless, the intracellular calcium increase induced by the FSH in Sertoli cells [30,31,40] could explain this difference. Fig. 1. Dose-dependent effect of FSH on glypican-1, syndecan-1 and syndecan-4 mRNAs expression. Sertoli cells from 20-day-old rats were incubated for 24 h in the presence of increasing concentrations (0–200 ngÆmL )1 ) of FSH. Total RNA was extracted as described in Material and methods. Then RNA (500 ng) was reverse transcribed and amplified by relative quantitative RT-PCR as described previously [24]. Glyp-1, Glypican-1; Synd-1, Syndecan-1; Synd-4, syndecan-4. (A) Agarose gels of one representative experiment. (B) Densitometry data are representative of five different experiments (mean ± SE). Ó FEBS 2002 FSH regulation of HSPG expression (Eur. J. Biochem. 269) 3463 Effect of intracellular calcium increase The possible involvement of intracellular calcium on the HSPG mRNAs expression was studied by using verapamil, an inhibitor of the calcium type L channels. Sertoli cell cultures were treated with 100 l M verapamil during 24 h. Figure 5 indicated that glypican-1 and syndecan-1 mRNAs expression was upregulated (+42 and +28%, respectively), whereas syndecan-4 mRNA was not. Moreover, Sertoli cell cultures were performed in the presence of EGTA (1.06 m M ), which chelates extracellular calcium and reduces its availability. EGTA upregulated glypican-1 and syndecan-1 mRNA expression as verapamil did (data not shown). Thus, the increase of glypican-1 and syndecan-1 mRNAs expression resulted from an impaired calcium influx. Our attempts to increase intracellular calcium levels, either by adding exogenous calcium or by using calcium ionophores (ionomycin or A23187) proved unsuccessful. At concentrations commonly used in the literature, these molecules led to subsequent cell death in our culture conditions and lower concentrations are inefficient in modifying Sertoli cell proteoglycan synthesis. Thus, the increase of intracellular calcium was appreci- ated indirectly by incubating Sertoli cell cultures with both FSH (100 ngÆmL )1 )andH8(5 l M ), a specific protein kinase A inhibitor. Figure 6 indicates that glypican-1 and synde- can-1 mRNAs expression was downregulated by the resulting intracellular calcium increase ()26 and )30%, respectively), whereas syndecan-4 mRNA was not. Thus, the effect of the increased intracellular calcium level on HSPG mRNA expression confirmed the results obtained with verapamil and EGTA. Our results suggest that: (a) the increase of intracellular cAMP and intracellular calcium levels contributes similarly to the FSH-induced inhibition of glypican-1 mRNA expression, whereas (b) the absence of FSH on syndecan-1 mRNA expression results from an antagonistic effect of increased intracellular cAMP and intracellular calcium levels. FSH, cAMP and intracellular calcium effects on glypican-1 and syndecans-1 and -4 mRNA expression during Sertoli cell development In Sertoli cells from 10-day-old rats, FSH (100 ngÆmL )1 ) downregulated the glypican-1 mRNA expression ()30%) whereas it did not modify syndecans mRNA expression (Table 1). The dbcAMP (1 m M ) induced the same effect as the hormone did. Indeed, the glypican-1 mRNA expression was inhibited ()44%) by dbcAMP (Table 1), whereas syndecans-1 and -4 mRNA expression was not affected (Tables 2 and 3). The intracellular calcium increase did not regulate HSPG mRNA expression at this age (Tables 1, 2 and 3). In Sertoli cells from 30-day-old rats, FSH (100 ngÆmL )1 ) and dbcAMP (1 m M ) inhibited the glypican-1 mRNA Fig. 2. dbcAMP action on glypican-1, syndecan-1 and syndecan-4 mRNAs expression. Sertoli cells from 20-day-old rats were incubated for 24 h in the presence (+) or in the absence (–) of 1 m M dbcAMP. Total RNA was extracted as described in Materials and methods. Then RNA (500 ng) was reverse transcribed and amplified by relative quantitative RT-PCR as described previously [24]. Glyp-1, glypican-1; synd-1, syndecan-1; synd-4, syndecan-4. (A) Agarose gel of one rep- resentative experiment. (B) Densitometry data are representative of three different experiments (mean ± SE). Each relative HSPG mRNA level under treatment is expressed vs. control which is arbi- trarily set to 100%. Fig. 3. Effect of cholera toxin on mRNAs expression. Sertoli cells from 20-days-old rats were incubated in the presence (+) or in the absence (–)of10lgÆmL )1 cholera toxin (CT) during 24 h. Total RNA was extracted as described in Materials and methods. Then, RNA (500 ng) was reverse transcribed and amplified by relative quantitative RT-PCR as described previously [24]. Glyp-1, glypican-1; synd-1, syndecan-1; synd-4, syndecan-4. (A) Agarose gel of one representative experiment. (B) Densitometry data are representative of seven different experi- ments (mean ± SE). Each relative HSPG mRNA level under treat- ment is expressed vs. control which is arbitrarily set to 100%. 3464 S. Brucato et al. (Eur. J. Biochem. 269) Ó FEBS 2002 expression by )33 and )30%, respectively (Table 1), whereas intracellular calcium increase did not influence glypican-1, syndecans-1 and -4 mRNAs expression. In contrast, FSH and dbcAMP upregulated the syndecan-1 and syndecan-4 mRNA expression in 30-days-old rat Sertoli cells (Tables 2 and 3). Indeed, FSH increased syndecan-1 and syndecan-4 mRNAs expression by +40 and +53%, respectively, and dbcAMP stimulated them by +35 and +55%, respectively (Tables 2 and 3). DISCUSSION This report shows, for the first time, the FSH regulation of HSPG mRNA expression in rat Sertoli cells. The effects of FSH, main effector of Sertoli cell functions, and its second messengers (increase in intracellular cyclic AMP and intracellular calcium levels) were evaluated on glypican-1, syndecan-1 and -4 mRNAs expression. Our data indicate the existence of a HSPG-specific regulation depending on the Sertoli cell developmental stage. FSH induces the inhibition of the glypican-1 mRNA expression in all studied Sertoli cell developmental stages (10, 20 and 30-days-old rats). In contrast, syndecan-1 and -4 mRNAs expression was not modified by this gonadotropin. The increase of intracellular cAMP level similarly reduced glypican-1 mRNA expression whatever Sertoli cell devel- opmental stage. Nevertheless, although syndecan-1 mRNA expression was not modified in Sertoli cells from 10-day-old rats, it was upregulated in 20- and 30-day-old rat Sertoli cells by this second messenger. Moreover, syndecan-4 mRNA expression was stimulated by intracellular cAMP increase in 30-day-old rat Sertoli cells. Until now, there has been little information concerning cAMP effects on cell surface HSPG mRNA expression. In NIH 3T3 fibroblasts, bFGF increases the transcription of the syndecan-1 gene by activating a bFGF-inducible response element (FiRE) present on syndecan-1 gene. It has been reported that the activation of FiRE by bFGF requires active PKA [41]. Although the syndecan mRNA expression is induced by PKA activation, the increase of syndecan mRNA expression results from increased intra- cellular cAMP level in Sertoli cells whereas the total cellular cAMP concentration was not implied on its increase in NIH 3T3 fibroblasts [41]. In vascular smooth muscle (VSM) cells, carbacyclin and forskolin, agents that elevate cAMP levels, failed to increase syndecan mRNA levels in contrast to our results [42]. Moreover, endothelin, which reduces cAMP accumulation by inhibiting adenylyl cyclase [43], also had no effect on HSPG expression. These data suggested that regulation of syndecan-1 expression occurred by cAMP-independent mechanisms in VSM cells. These results and our work indicate that the mechanisms respon- sible for regulating the synthesis of these HSPG are complex and that cAMP effect could be cell type-dependent. Fig. 5. Effect of verapamil on glypican-1, syndecans-1 and )4 mRNAs expression. Sertoli cells from 20-day-old rats were incubated in the presence (+) or in the absence (–) of 100 l M verapamil during 24 h. Total RNA was extracted as described in Materials and methods. Then, RNA (500 ng) was reverse transcribed and amplified by relative quantitative RT-PCR as described previously in [24]. Glyp-1, glypican-1; synd-1, syndecan-1; synd-4, syndecan-4. (A) Agarose gel of one representative experiment. (B) Densitometry data are representa- tive of seven different experiments (mean ± SE). Each relative HSPG mRNA level under treatment is expressed vs. control which is arbi- trarily set to 100%. Fig. 4. Action of a cAMP phosphodiesterase inhibitor, RO-20-1724 on mRNAs expression. Sertoli cells from 20-day-old rats were incubated in the presence (+) or in the absence (–) of 250 l M RO-20-1724 during 24 h. Total RNA was extracted as described in Material and Methods. Then, RNA (500 ng) was reverse transcribed and amplified by relative quantitative RT-PCR as described previously in [24]. Glyp-1, glypican-1; synd-1, syndecan-1; synd-4, syndecan-4. (A) Agarose gel of one representative experiment. (B) Densitometry data are representa- tive of three different experiments (mean ± SE). Each relative HSPG mRNA level under treatment is expressed vs. control which is arbi- trarily set to 100%. Ó FEBS 2002 FSH regulation of HSPG expression (Eur. J. Biochem. 269) 3465 The FSH-binding to Sertoli cells activates the cAMP- dependent protein kinase A signaling pathway, resulting in the phosphorylation and activation of transcription factors such as CREB (cAMP response element binding protein), CREM (cAMP response element modulator), ATF-1 and AP-2 [44–46]. The transcription factor AP-2 binds to a consensus binding site in glypican-1, syndecans-1 and -4 promoters [47–50]. AP-2 could be potentially implicated in the HSPG mRNA expression regulation. Moreover, AP-2 sites are a common target of PKA and PKC signaling pathways [51]. Therefore, this regulation is different depending on PKA (data not shown) 3 or PKC activation [24]. Thus, other PKA- and PKC-inducible elements that remain to be elucided could explain the observed differential regulation. Beyond increased cAMP synthesis, intracellular calcium increase is also involved in signal transduction triggered by FSH [30,31,52]. Our data demonstrate that L-type voltage- operated calcium channel blocker, verapamil, induces the increase of glypican-1 and syndecan-1 mRNAs expression in Sertoli cells from 20-day-old rats. The effect of verapamil on HSPG mRNA expression probably results from the decrease of transmembrane calcium influx. Although no attempt was made to measure intracellular calcium concen- tration, the above hypothesis is supported by (a) a similar effect of EGTA and (b) the action of both FSH and H8, a specific PKA inhibitor. In this second case, the resulting increase in intracellular calcium down regulates the glypi- can-1 and syndecan-1 mRNAs expression leading to the same conclusion concerning the calcium effect. Fig. 6. Action of H8, a specific inhibitor of protein kinase A. Sertoli cells from 20-day-old rats were incubated without (–) or with (+) FSH (100 ngÆmL )1 ) or in combination of FSH (100 ngÆmL )1 )andH8 (5 l M ) for 24 h. Total RNA was extracted as described in Materials and methods. Then, RNA (500 ng) was reverse transcribed and amplified by relative quantitative RT-PCR as described in [24]. Glyp-1, glypican-1; synd-1, syndecan-1; synd-4, syndecan-4. (A) Agarose gel of one representative experiment. (B) Densitometry data are representa- tive of three different experiments (mean ± SE). Each relative HSPG mRNA level under treatment is expressed vs. control which is arbi- trarily set to 100%. Table 1. FSH, cAMP and calcium increase effects on glypican-1 mRNA expression during Sertoli cells development. Sertoli cells from 10-, 20- and 30-day old rats were incubated for 24 h with 100 ngÆmL )1 FSH, 1 mM dbcAMP (intracellular cAMP increase), or 100 ngÆmL )1 FSH plus 5 l M H8 (increase in intracellular calcium). Each relative HSPG mRNA level under treatment is expressed versus control which is arbitrarily set to 100%. Each percentage is obtained from densitometry data representative of at least three different experiments (mean ± SE). *, Significant values. Glypican-1 mRNA expression relative to control (%) Sertoli cells 10-days-old Sertoli cells 20-day-old Sertoli cells 30-day-old FSH )30 ± 2* )45 ± 3* )33 ± 4* dbcAMP )44 ± 3* )56 ± 5* )30 ± 2* Calcium )9±2 )26 ± 2* )3±2 Table 2. FSH, cAMP and calcium increase effects on syndecan-1 mRNA expression during Sertoli cells development. Sertoli cells from 10, 20 and 30-day-old rats were incubated for 24 h with 100 ngÆmL )1 FSH, 1 m M dbcAMP (intracellular cAMP increase), or 100 ngÆmL )1 FSH plus 5 l M H8 (increase in intracellular calcium). Each relative HSPG mRNA level under treatment is expressed versus control which is arbitrarily set to 100%. Each percentage is obtained from densi- tometry data representative of at least three different experiments (mean ± SE). *, Significant values. Glypican-1 mRNA expression relative to control (%) Sertoli cells 10-days-old Sertoli cells 20-day-old Sertoli cells 30-day-old FSH +10 ± 4 +3 ± 1 +40 ± 3* dbcAMP )4 ± 3 +50 ± 5* )35 ±5* Calcium )5±3 )30 ± 2* )6±3 Table 3. FSH, cAMP and calcium increase effects on syndecan-4 mRNA expression during Sertoli cells development. Sertoli cells from 10, 20 and 30-day-old rats were incubated for 24 h with 100 ngÆmL )1 FSH, 1 m M dbcAMP (intracellular cAMP increase), or 100 ngÆmL )1 FSH plus 5 l M H8 (increase in intracellular calcium). Each relative HSPG mRNA level under treatment is expressed versus control which is arbitrarily set to 100%. Each percentage is obtained from densi- tometry data representative of at least three different experiments (mean ± SE). *, Significant values. Glypican-1 mRNA expression relative to control (%) Sertoli cells 10-days-old Sertoli cells 20-day-old Sertoli cells 30-day-old F S H +8 ± 3 +3 ± 1 +5 3 ± 4 * dbcAMP )3±2 +1±5 )55 ± 6* Calcium )4±1 )4±2 )11 ± 3 3466 S. Brucato et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Thus, FSH, as the increase in intracellular cAMP and intracellular calcium, decreases the glypican-1 mRNA expression in Sertoli cells from 20-day-old rats. On the other hand, FSH-stimulated syndecan-1 mRNA expression is not modulated as it results from the antagonistic effects of increased intracellular cAMP and intracellular calcium levels. Moreover, calcium induces no effect on glypican-1 and syndecan-1 mRNAs expression in 10- and 30-day-old rat Sertoli cells. Finally, syndecan-4 mRNA expression is not regulated by this pathway in all studied Sertoli cell developmental stages. Until now, there has been little data about calcium regulation on HSPG mRNA expression in Sertoli cells and other cell systems. In cultured Sertoli cells from 20-day-old rats, verapamil and EGTA induced a sharp decrease in proteoglycan synthesis, affecting both secreted and cell- associated proteoglycans [53]. Intracellular calcium concen- tration either stimulates proteoglycan synthesis in bovine granulosa cells [54,55] and in breast cancer cells [56] or decreases proteoglycan synthesis in chondrocytes [57] and parathyroid cells [58,59]. In vascular smooth muscle cells, Cizmeci-Smith & Carey [60] demonstrated that calcium is required for syndecan-1 mRNA expression but that changes in intracellular calcium concentrations alone are not suffi- cient to induce syndecan expression. Further experiments will be necessary to understand calcium regulation on glypican-1 and syndecans-1 and -4 in rat Sertoli cell development. The physiological significance of the FSH regulation of glypican-1 and syndecans-1 and -4 mRNA remains to be elucidated. FSH stimulates the postnatal and pubertal development of Sertoli cells [61]. This age dependency is described for all FSH-stimulated intracellular events in isolated Sertoli cells [62–64]. Thus, increased cAMP but also inhibition of phosphodiesterase, activation of protein kin- ase, RNA and protein synthesis or mitotic activity present a peak of activity around 20 days of age which corresponds to the tight junctions formation between in vivo Sertoli cells [63,65]. During Sertoli cell ontogenesis, the lack of FSH responsiveness could be the consequence of cAMP inactivity by phosphodiesterase activity increase [19] rather than a reduced FSH receptors number as these receptors are increased in the same time. Phamanthu et al. [23] suggests a possible involvement of cell HSPG in the age- related increases in Sertoli cell phosphodiesterase activity. The increase of syndecans-1 and -4 expression induced by FSH in 30-day-old rat Sertoli cells suggested that these proteoglycans may be positive regulators of phosphodi- esterase activity. Indeed, the syndecan-4 cytoplasmic domain binds and regulates the PKC-a activity [66,67]. Considering these data, syndecans would regulate enzy- matic activities confined in the plasma membrane. The mechanism by which syndecans could increase phospho- diesterases activity is still unknown. The presence of a hydrophobic domain in the phosphodiesterase structure would suggest a possible insertion in the membrane [68]. Cyclic AMP-PDE activity is found associated with both cytosol and membrane fraction [39,68,69]. The mechanism whereby various PDEs are targeted to particular mem- brane sites, or occur in the cytosol, and the functional significance for specific intracellular locations of PDE is not understood. Thus, it is tempting to speculate that high concentrations of syndecans in the plasma membrane lead to changes in the membrane architecture, thus reducing association of one or more PDE isoforms with the cell membrane and, consequently, their catalytic properties towards cAMP. REFERENCES 1. David, G. (1993) Integral membrane heparan sulphate proteo- glycans. FASEB J. 7, 1023–1030. 2. Kim, C.W., Goldberger, O.A., Gallo, R.L. & Bernfield, M. (1994) Members of syndecans family of heparan sulfate proteoglycans are expressed in distinct cell-, tissue-, and specific pattern. Mol. Biol. Cell 5, 797–805. 3. Paine-Saunders, S., Viviano, B.L. & Saunders, S. (1999) GPC6,a novel member of the glypican gene family, encodes a product structurally related to GPC4 and is colocalized with GPC5 on human chromosome 13. Genomics 57, 455–458. 4. Rapraeger, A., Jalkanen, M., Endo, E., Koda, J. & Bernfield, M. (1985) The cell surface proteoglycan from mouse mammary epi- thelial cells bear chondroitine sulfate and heparan sulfate glyco- saminoglycans. J. Biol. Chem. 260, 11046–11052. 5. Hinkes, M.T., Goldberger, O.A., Neumann, P.E., Kokenyesi, R. & Bernfield, M. (1993) Organisation and promoter activity of the mouse syndecan-1 gene. J. Biol. Chem. 268, 11440–11448. 6. Watanabe, K., Yamada, H. & Yamaguchi, Y. (1995) K-glypican: a novel GPI-anchored heparan sulphate proteoglycan that is highly expressed in developing brain and kidney. J. Cell Biol. 130, 1207–1218. 7. Pilia, G., Hughes-Benzies, R.M., Mackenzie, A., Baybayan, P., Chen,E.Y.,Huber,R.G.,Cao,A.,Forabosco,A.&Schliessinger, D. (1996) Mutations in GPC3, a glypican gene, cause the simpson-Gobali-Behmel overgrowth syndrome. Nat. Genet. 12, 241–247. 8. Carey, D.J. (1997) Syndecans, multifunctional cell-surface co-receptors. Biochem. J. 327, 1–16. 9. Rapraeger, A.C. (2001) Molecular interactions of syndecans during development. Cell Dev. Biol. 12, 107–116. 10. De Cat, B., David, G. (2001) Developmental roles of the glypi- cans. Cell Dev. Biol. 12, 117–125. 11. Romaris, M., Bassols, A. & David, G. (1995) Effect of trans- forming growth factor-beta 1 and basic fibroblast growth factor on the expression of cell surface proteoglycans in human lung fibroblasts. Enhanced glycanation and fibronectin-binding of CD44 proteoglycan, and down-regulation of glypican. Biochem. J. 310, 73–81. 12. Bansal, R., Kumar, R., Murray, K. & Pfeiffer, S.E. (1996) Developmental and FGF-2 mediated regulation of syndecans (1–4) and glypican in oligodendrocytes. Mol. Cell. Neurosci. 7, 276–288. 13. Elenius, K., Maatta, A., Salmivirta, M. & Jalkanen, M. (1992) Growth factors induce 3T3 cells to express bFGF-binding syn- decan. J. Biol. Chem. 267, 6435–6441. 14. Sebestye ´ n,A.,Gallai,M.,Knittel,T.,Ambrust,T.,Ramadori,G. & Kovalszky, I. (2000) Cytokine regulation of syndecan expres- sion in cells of liver origin. Cytokine 12, 1557–1560. 15. Bernfield, M., Go ¨ tte, M., Park, P.W., Reizes, O., Fitzgerald, M.L., Lincecum, J. & Zako, M. (1999) Functions of cell surface heparan sulfate proteoglycans. Annu. Rev. Biochem. 68, 729–777. 16.Carey,D.J.,Stahl,R.C.,Cizmeci-Smith,G.&Asundi,V.K. (1994) Syndecan-1 expressed in Shawnn cells causes morphologi- cal transformation and cytoskeletal reorganization and associates with actin during cell spreading. J. Cell Biol. 124, 161–170. 17. Saoncella, S., Echtermeyer, F., Denhez, F., Nowlen, J.K., Mosher, D., Robinson, S.D., Hynes, R.O. & Goetinck, P.F. (1999) Syn- decan-4 signals cooperatively with integrins in a Rho-dependent manner in the assembly of focal adhesions and actin stress fibers. Proc. Natl Acad. Sci. USA 96, 2805–2810. Ó FEBS 2002 FSH regulation of HSPG expression (Eur. J. Biochem. 269) 3467 18. Oh, E.S., Woods, A. & Couchman, J.R. (1997) Syndecan-4 pro- teoglycan regulates the distribution and activity of protein kinase C. J. Biol. Chem. 272, 8133–8136. 19. Griswold, M.D. (1998) The central role of Sertoli cells in sper- matogenesis. Cell Dev. Biol. 9, 411–416. 20. Mounis, A., Barbey, P., Langris, M. & Bocquet, J. (1991) Detergent-solubilized proteoglycans in rat testicular Sertoli cells. Biochim. Biophys. Acta 1074, 424–432. 21. Brucato, S., Fagnen, G., Villers, C., Bonnamy, P.J., Langris, M. & Bocquet, J. (2001) Biochemical characterization of integral mem- brane heparan sulfate proteoglycans in Sertoli cells from immature rat testis. Biochim. Biophys. Acta 1510, 474–487. 22. Phamantu, N.T., Bonnamy, P.J., Bouakka, M. & Bocquet, J. (1995) Inhibition of proteoglycan synthesis induces an increase in follicle stimuling hormone (FSH)-stimulated estradiol produc- tion by immature rat Sertoli cells. Mol. Cell Endocrinol. 109, 37–45. 23. Phamantu, N.T., Fagnen, G., Godard, F., Bocquet, J. & Bonnamy, P.J. (1999) Sodium chlorate induces undersulfation of cellular proteoglycans and increases in FSH-stimulated estradiol produc- tion in immature rat Sertoli cells. J. Androl. 20, 241–250. 24. Brucato, S., Harduin-Lepers, A., Godard, F., Bocquet, J. & Villers, C. (2000) Expression of glypican-1, syndecan-1 and syn- decan-4 mRNAs protein kinase C-regulated in rat immature Sertoli cells by semi-quantitative RT-PCR analysis. Biochim. Biophys. Acta. 1474, 31–40. 25. Brucato, S., Bocquet, J. & Villers, C. (2002) Cell surface heparan sulfate proteoglycans: target and partners of the basic fibroblast growth factor in rat Sertoli cells. Eur. J. Biochem. 269, 502–511. 26. Skinner, M.K. & Fritz, I.B. (1985) Structural characterization of proteoglycans produced by testicular peritubular cells and Sertoli cells. J. Biol. Chem. 260, 11874–11883. 27. Leung, P.C.K. & Steele, G.I. (1992) Intracellular signaling in the gonads. Endocrinol. Rev. 13, 476–498. 28. Antoni, F.A. (2000) Molecular diversity of cyclic AMP signalling. Front Neuroendocrinol. 21, 103–132. 29. Means, A.R., Dedman, J.R., Tash, J.S., Tindall, D.J., vanSickle, M. & Welsh, M.J. (1980) Regulation of the testis Sertoli cell by follicle stimulating hormone. Ann. Rev. Physiol. 42, 59–70. 30. Grasso, P. & Reichert Jr, L.E. (1989) Follicle-stimulating hormone receptor-mediated uptake of 45 Ca ++ by proteoliposomes and cultured rat Sertoli cells: evidence for involvment of voltage-acti- vated and voltage-independent calcium channels. Endocrinol. 125, 3029–3036. 31. Gorczynska, E. & Handelsman, D.J. (1991) The role of calcium in follicle-stimulating hormone signal transduction in Sertoli cells. J. Biol. Chem. 266, 23739–23744. 32. D’Agostino, A., Mene, P. & Stefanini, M. (1992) Voltage-gated calcium channels in rat Sertoli cells. Biol. Reprod. 46, 414–418. 33. Taranta, A., Morena, A.R., Barbacci, E. & D’agostino, A. (1997) x-Conotoxin-sensitive Ca 2+ voltage-gated channels modulate proteinsecretioninculturedratSertolicells.Mol. Cell. Endocrinol. 126, 117–123. 34. Tung, P.S., Skinner, M.K. & Fritz, I.B. (1984) Fibronectin synthesis is a marker for peritubular contaminants in Sertoli cell- enriched cultures. Biol. Reprod. 30, 199–211. 35. Galdieri, M., Ziparo, E., Palombi, F., Russo, M.A., & Stefanini, M. (1981) Pure Sertoli cell cultures: a new model for the Study of somatic-germ cell interactions. J. Androl. 2, 249–254. 36. Chomczynski, P. & Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159. 37. West, D.C., Sattar, A. & Kumar, S. (1985) A simplified in situ solubilization procedure for the determination of DNA and cell number in tissue cultured mammalian cells. Anal. Biochem. 147, 289–295. 38.Fritz,I.B.,Griswold,M.D.,Louis,B.G.&Dorrington,J.H. (1976) Similarity of responses of cultured Sertoli cells to cholera toxin and FSH. Mol. Cell. Endocrinol. 5, 286–294. 39. Conti, M., Nemoz, G., Sette, C. & Vicini, E. (1995) Recent pro- gress in understanding the hormonal regulation of phosphodies- terases. Endocrine Rev. 16, 370–389. 40. Sharma, O.P., Flores, J.A., Leong, D.A. & Veldhuis, J.D. (1994) Cellular basis for follicle-stimulating hormone-stimulated calcium signaling in rat Sertoli cells: possible dissociation from effects of adenosine 3¢,5¢-monophosphate. Endocrinol. 134, 1915–1923. 41. Pursiheimo, J.P., Jalkanen, M., Tasken, K. & Jaakkola, P. (2000) Involvement of protein kinase A in fibroblast growth factor- 2-activated transcription. Proc.NatlAcad.SciUSA97, 168–173. 42. Cizmeci-Smith, G., Stahl, R.C., Showalter, L.J. & Carey, D.J. (1993) Differential expression of transmembrane proteoglycans in vascular smooth muscle cells. J. Biol. Chem. 268, 18740–11877. 43. Hilal-Dandan, R., Urasawa, K. & Brunton, L.L. (1992) Endo- thelin inhibits adenylate cyclase and stimulates phosphoinositide hydrolysis in adult cardiac myocytes. J. Biol. Chem. 267, 10620– 10624. 44. Daniel, P.B., Walker, W.H. & Habener, J.F. (1998) Cyclic AMP signaling and gene regulation. Annu. Rev. Nutr. 18, 353–383. 45. Walker, W.H., Daniel, P.B. & Habener, J.F. (1998) Inducible cAMP early repressor ICR down-regulation of CREB gene expression in Sertoli cells. Mol. Cell. Endocrinol. 143, 167–178. 46. Gronning, L.M., Dahle, M.K., Tasken, K.A., Enerback, S., Hedin, L., Tasken, K. & Knutsen, H. (1999) Isoform-specific regulation of the CCAAT/enhancer-binding protein family of transcription factors by 3¢,5¢ cyclic adenosine monophsphate in sertoli cells. Endocrinology 140, 835–843. 47. Takagi, A., Kojima, T., Tsuzuki, S., Katsumi, A., Yamazaki, T., Sugiura, I., Hamaguchi, M. & Saito, H. (1996) Structural orga- nization and promoter activity of the Human ryudocan gene. J. Biochem. 119, 979–984. 48. Tsuzuki,S.,Kojima,T.,Katsumi,A.,Yamazaki,T.,Sugiura,I.& Saito, H. (1997) Molecular cloning, genomic organization, pro- moter activity, and tissue-specific expression of the mouse ryu- docan gene. Biochem. J. 122, 17–24. 49. Asundi, V.K., Keister, B.F. & Carey, D.J. (1998) Organization, 5¢-flanking sequence and promoter activity of the rat GPC1 gene. Gene 206, 255–261. 50. Maatta, A., Jaakkola, P. & Jalkanen, M. (1999) Extracellular matrix-dependent activation of syndecan-1 expression in kerati- nocyte growth factor-treated keratinocytes. J. Biol. Chem. 274, 9891–9898. 51. Imagawa, M., Chiu, R. & Karin, M. (1987) Transcription factor AP-2 mediates induction by two different signal-transduction pathways: protein kinase C and cAMP. Cell 51, 251. 52. Grasso, P. & Reichert Jr, L.E. (1990) Follicle-stimulating hormone receptor-mediated uptake of Ca ++ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin- sensitive guanine nucleotide binding proteins or adenylate cyclase. Endocrinol. 127, 949–956. 53. Fagnen, G., Phamantu, N.T., Bocquet, J. & Bonnamy, P.J. (1999) Inhibition of transmembrane calcium influx induces decrease in proteoglycan synthesis in immature rat Sertoli cells. J. Cell. Bio- chem. 76, 322–331. 54.Lenz,R.W.,Ax,R.L.&First,N.L.(1982)Proteoglycanpro- duction by bovine granulosa cells in vitro is regulated by calmo- dulin and calcium. Endocrinology 110, 1052–1054. 55. Bellin, M.E., Lenz, R.W., Steadman, L.E. & Ax, R.L. (1983) Proteoglycan production by bovine granulosa cells in vitro occurs in response to FSH. Mol. Cell. Endocrinol. 29, 51–65. 56. Vandewalle, B., Revillion, F., Hornez, L. & Lefebvre, J. (1994) Calcium regulation of heparan sulfate proteoglycans in breast cancer cells. J. Cancer Res. Clin. Oncol. 120, 389–392. 3468 S. Brucato et al. (Eur. J. Biochem. 269) Ó FEBS 2002 57. Eilam, Y., Beit-Or, A. & Nevo, Z. (1985) Decrease in cytosolic free Ca 2+ and enhanced proteoglycan synthesis induced by cartilage derived growth factors in cultured chondrocytes. Biochem. Bio- phys. Res. Commun. 132, 770–779. 58. Takeuchi, Y., Sakagushi, K., Yanagishita, M., Aurbach, G.D. & Hascall, V.C. (1990) Extracellular calcium regulates distribution and transport of heparan sulfate proteoglycans in rat parathyroid cell line. J. Biol. Chem. 265, 13661–13668. 59. Muresan, Z. & MacGregor, R. (1994) The release of parathyroid hormone and the exocytosis of a proteoglycan are modulated by extracellular Ca 2+ in a similar manner. Mol. Biol. Cell 5, 725–737. 60. Cizmeci-Smith, G. & Carey, D.J. (1997) Thrombin stimulates syndecan-1 promotor activity and expression of a form of syn- decan-1 that binds antithrombin III in vasular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 17, 2609–2616. 61. Gondos, B. & Berndston, W.E. (1993) Postnatal and pubertal development. In The Sertoli Cell (par Russel, L.D. & Griswold, M.D., eds), pp. 115–154. Cache River Press, USA. 62. Fritz, I.B. (1979) In Biochemical actions of hormones (Litwack, E., ed.), pp. 249–281. Academic Press, New York. 63. Means, A.R., Dedman, J.R., Fakunding, J.L. & Tindall, D.J. (1978) In Receptors and Hormone Action (Birnbaumer, L. & O’Malley, B.W., eds), pp. 363–393. Academic Press, New York. 64. Means, A.R., Dedman, J.R., Welsh, M.J., Marcum, M. & Brinkley, B.R. (1979) In Ontogeny of Receptors and Reproductive Hormone Action (Hamilton,T.,Clark,J.&Sadler,W.,eds), pp. 207–224. Raven, New York. 65. Gilula, N.B., Fawcett, D.W. & Aoki, A. (1976) The Sertoli cell occluding junctions and gap junctions in mature and developing mammalian testis. Dev. Biol. 50, 142–168. 66. Oh, E.S., Woods, A., Lim, S.T., Theibert, A.W. & Couchman, J.R. (1998) Syndecan-4 proteoglycan cytoplasmic domain and phosphatidylinositol 4,5 biphosphate coordinately regulate pro- tein kinase C activity. J. Biol. Chem. 273, 10624–10269. 67. Horowitz, A. & Simons, M. (1998) Phosphorylation of the cyto- plasmic tail of syndecan-4 regulates activation of protein kinase Ca. J. Biol. Chem. 273, 25548–25551. 68. Shakur, Y., Pryde, J.G. & Houslay, M.D. (1993) Engineered deletion of the unique N-terminal domain of the cyclic AMP- specific phosphodiesterase RD1 prevents plasma membrane association and the attainment of enhanced thermostability without altering its sensitivity to inhibition by rolipram. Biochem. J. 292, 677–688. 69. Soderling, S.H. & Beavo, J.A. (2000) Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions. Curr. Opin. Cell Biol. 12, 174–179. Ó FEBS 2002 FSH regulation of HSPG expression (Eur. J. Biochem. 269) 3469 . Regulation of glypican-1, syndecan-1 and syndecan-4 mRNAs expression by follicle-stimulating hormone, cAMP increase and calcium influx during rat Sertoli. m M of dbcAMP and maintained for high concentrations of dbcAMP. The syndecan-1 mRNA expression was increased by 1 m M of dbcAMP (+50%) whereas the syndecan-4