Inhibitory effect of bFGF on FSH-stimulated estradiol synthesis by Sertoli cells cultured in the presence of Ro-20-1724, a cAMP phos- phodiesterase inhibitor.. bFGF effect on Sertoli ce
Trang 1Cell surface heparan sulfate proteoglycans
Target and partners of the basic fibroblast growth factor in rat Sertoli cells
Sylvie Brucato, Jean Bocquet and Corinne Villers
Laboratoire de Biochimie, IRBA, Université de Caen, France
Basic fibroblast growth factor (bFGF) regulates diversified
biological functions in rat Sertoli cells This report demon-
strates that bFGF inhibits steroidogenesis in developing rat
Sertoli cells Follicle stimulating hormone (FSH)-stimulated
estradiol production was reduced by bFGF Moreover, the
amount of cytochrome P450 aromatase, responsible for the
irreversible transformation of androgens into estrogens, is
decreased by bFGF at the transcriptional level The bFGF
inhibitory effect was also observed in the presence of dibu-
tyryl-cAMP, cholera toxin or RO-20-1724, all inducing high
levels of cAMP, the second messenger of FSH
Heparan sulfate proteoglycans (HSPGs) were shown to be
required as cofactors for bFGF signaling Indeed, sodium
chlorate, described to drastically decrease proteoglycan sul- fation, abolishes the bFGF downregulation of FSH-stimu- lated estradiol synthesis previously observed Glypican-1, syndecan-1 and -4, potential bFGF coreceptors, are mainly regulated at the transcriptional level This report shows that the bFGF regulation of their expression specifically 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 fibroblast growth factor (bFGF or FGF-2)
belongs to a large FGF family of 21 structurally related
members [1] This growth factor is produced by many cell
types and tissues, including testis [2] Its biological activity is
pleiotropic [3] as it influences 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 steroidogenesis [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 affinity cell surface bFGF receptors (FGFR-1
to FGFR-4) [10] In addition, bFGF binds to heparan
sulfate proteoglycans (HSPG) on the cell surface [11]
Oligosaccharidic sequences of HS chains are defined for the
bFGF binding and for the recognition of the specific bFGF
receptor, leading to the formation of a ternary complex
comprising HSPG-bFGF-FGER These oligosaccharidic
motifs are differently sulfated related to the synthesis
pathway itself and depending on the cell type The resulting
Correspondence to 8 Brucato, Laboratoire de Biochimie, IRBA,
Université de Caen, Esplanade de la Paix, 14032 Caen cedex, France
Fax: + 33 2 31 95 49 40, Tel.: + 33 2 31 56 65 76,
E-mail: s_brucato@yahoo.fr
Abbreviations: bFGF, basic fibroblast 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 modified Eagle’s medium; RhFGF, recombinant human basic
FGF; PAPS, phosphoadenosine phosphosulfate; AMV, avian myelo-
blastosis virus
(Received 30 May 2001, revised 1 October 2001, accepted 14
November 2001)
structural microheterogeneity modulates bFGF affinity 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 significantly reduced when compared to cells expressing HSPG [4,1 1-15]
Sertoli cells are the principal source of estradiol produc-
tion in the immature testis [16,17] Significant estrogen
synthesis is present in Sertoli cells of early postnatal rats, with a sharp reduction during subsequent maturation [18,19]
The present work firstly aims to evaluate the effect of bFGF on follicle stimuling hormone (FSH)-estradiol syn- thesis and cytochrome P450 aromatase mRNA expression
in 20 days old-rat Sertoli cells The involvement of the cAMP pathway was evaluated using three approaches, all inducing differently high levels of cAMP: (a) dibutyryl cyclic AMP (dbcAMP), a structural analogue of cAMP; (b) cholera toxin, a protein Gs activator; and (c) RO-20-1724, a specific phosphodiesterase inhibitor
Then, we investigated bFGF effect on FSH-estradiol synthesis in the absence of HSPG in 20-day-old-rat Sertoli 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 expressed [22] More-
over, syndecan-1 [23], syndecan-4 [24] and glypican-1 [25] are potential coreceptors of bFGF, and are essentially 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
Trang 2specifically upregulated by PKC-activation in contrast to
syndecan-4 transcription [22] Until now, nothing has been
known about the bFGF regulation of glypican-1 and
syndecan-| and -4 mRNA expression in developing Sertoli
cells Our present study demonstrates that bFGF influences
this expression in 20-day-old-rat Sertoli cells
During testicular development, the physiology of Sertoli
cells is modified 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 Therefore, 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 FSH (oFSH-21) was kindly provided by the National
Institute of Arthritis, Metabolic and Digestive Diseases
(Pituitary Hormone Distribution program, Bethesda, MD,
USA) 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 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)2cA MP (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 buffer 5 x, oligo d(T) 15, dNTPs, RNasin,
AMV-reverse transcriptase, Thermus aquaticus (Taq) DNA
polymerase reaction buffer 10 x, Tag DNA polymerase and
MgCl were from Promega (Charbonntre-les-bains,
France) The oligonucleotide primers were synthesized and
purified by Eurobio (Les Ulis, France) Recombinant
human basic fibroblast growth factor (RhFGF) were from
R & D Systems (Abingdon, UK) 2,4,6,7[H]-17f estradiol
(3.77 TBqg:mmol') 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 rats from our
own colony were killed by cervical dislocation Sertoli cells
were obtained by sequential enzymatic digestion including
trypsin, collagenase and hyaluronidase as described previ-
ously [27]
Sertoli cells were seeded at the concentration of
250 000 cellscm ” in 24-well dishes or in 75-cm* plastic
flasksand cultured for48 hinHam's F12/DMEM(I : l,v/v)
supplemented with 2% Ultroser SF in order to attach the
Sertoli cells in a humidified atmosphere of 5% CO, 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 mm Tris/HCl (pH 7.4) [28] Sertoli cells were then cultured for two days in culture medium devoid of Ultroser before being used on day 5 after plating
For the aromatase assay, Sertoli cells were incubated for
24 h with testosterone (200 ngmL™'), oFSH (100 ngmL7') and/or bFGF (5 ngmL™') and/or sodium chlorate (10 mm)
Before RNA extraction, cells were incubated for 24 h either in the absence or in presence 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 Chomezynski & Sacchi [29] using Trizol reagent The integrity and quality of purified RNA were controlled by 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 reaction mixture containing the reaction buffer (50 mm Tris/HCl, pH 8.3, 50 mm KCI;
10 mm MgCh, 0.5 mm Spermidine, dithiothreitol 10 mm),
1 um oligo d(T);;, 500 um dNTPs, 20 UI RNasin, 18 UI AMV-reverse transcriptase in 20 nL final 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 first strand synthesis product (0.1 tg) was used as template to amplify each cDNA PCR was performed with 250 um dNTPs, Tag DNA polymerase reaction buffer (50 mm KCl, 10 mm Tris/HCl, pH 9; 0.1% Triton X-100), 2.5 UI Tag DNA polymerase, MgCl, 1.5mm, 10 pmol of each primer (Table 1) in a 20-unL reaction volume
The PCR was started at 94 °C 1 min and followed by up
to 27 cycles of amplification for the three proteoglycans and
20 cycles for the internal control, B-actin as described previously [23], which consisted of a denaturating step (at
94 °C for | min), an annealing step (at 55 °C for 1 min) and
an extension step (at 72°C for 2 min) then a final
elongation step (at 72 °C for 10 min) in ROBOCYCLER®
Gradient 40 (Stratagene)
The cytochrome P450 aromatase cDNA was amplified at
94 °C for 1 min for 30 cycles as described previously [30], which consisted of a denaturating step (at 94 °C for 1 min),
an annealing step (at 60 °C for 30 s) and an extension step (at 72 °C for 1 min) then a final elongation step (at 72 °C for 10 min) in ROBOCYCLER® Gradient 40 (Stratagene)
To check for contaminating genomic DNA, a RT-PCR was performed on RNA without AMV reverse transcriptase (data not shown) In all negative PCR control reactions, cDNA templates were replaced with sterile water to check the absence of contaminants
Aliquots (10 uL) of the PCR reaction were size- separated on a 4% agarose gel equilibrated in Tris/ acetate/EDTA (40 mm Tris/acetate, 1 mm EDTA) Gels were stained with ethidium bromide (1 ugmL”), photo- graphed using Polaroid film under UV light and ana- lysed using a AGFA SnapScan 1200” Scanner®, Adobe
Trang 3Table 1 Primers for PCR amplification
5’-802 CTCTTTGATGACAGAAGTGCCT-3’
5’-450 AAAAATGTTGCTGCCCTG-3’
S’-1054 CCTTTGAGCACATTTCGGCAA-3’
P450 aromatase S’-1555SGCTTCTCATCGCAGAGTATCCGG-3’ 289
S’-1821CAAGGGTAAATTCATTGGGCTTGG-3’
5-3222 AGCCATGCCAAATGTCTCAT-3/
PHOTOSHOP” software and the NIH IMAGE computer
program (http://rsb.info.nth.gov/nih-image)
Radio immuno assay of estradiol 17-B
Culture medium was extracted with 5 vol of diethylether
and estradiol was quantified by radiotmmunoassay using a
specific antibody purchased from Biosys (Compiegne,
France) The only significant cross reactions were for
2-methoxy-estradiol (5%), estradiol 17a (0.28%), estrone
and estriol (0.45%) The sensitivity of the assay was 6 pg per
tube Intra- and interassay coefficients of variation were less
than 10% The analysis of the radioimmunoassay data was
performed using the SECURIA program from the Packard
Instrument Company (Meriden, CT, USA)
DNA quantification
The DNA content of the cell layer at the end of incubation
was quantified by the method of West ef al [31] After
solubilization of the cell layer in 1 m NaOH and subsequent
neutralization by 1 M KH»,PO,, DNA was quantified in a
Kontron spectrofluorimeter using Hoescht 33258 as fluo-
rescent probe and calf thymus as standard
Statistical analysis
Allexperimental data were presented as the mean of duplicate
(estradiol) determinations of three wells in, at least, three
different cultures within each treatment group Results were
normalized in pg (estradiol) per 10° cells Statistical signifi-
cance between groups was determinated by Student’s paired
t-test Differences were considered significant 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 incubated for 24 h
with FSH (100 ngmL7') and increasing concentrations of
bFGF (0.1-10 ngmL~') 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 reached for 5 ng-mL' In
contrast, estradiol synthesis was not regulated by bFGF in
the absence of FSH (data not shown)
Direct implication of cAMP increase, the second mes- senger of FSH, in the bFGF regulation was evaluated by addition to the culture medium of either 1 mm dbcAMP or
10 jtg-mL' cholera toxin Their addition increased estradiol 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%, respectively) on estradiol synthesis as the one described in Fig 1 (Table 2)
The cAMP level elevation was also aprehended in the presence of 100 ngmL~' FSH and 250 um RO 20-1724, a specific inhibitor of cAMP-specific phosphodiesterase [32]
In these conditions, a significant increase (about + 65%) of estradiol production was obtained (Fig 2) as already described We observed that concomittant treatment with
5 ngmL~' 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
600
i oS oe
200
Control FSH FSH FSH FSH FSH (100ng/ml) +bFGF +bFGF +bFGF +bFGF
Fig 1 Dose-related effect of bFGF on FSH-stimulated estradiol syn- thesis in immature cultured rat Sertoli cells Sertoli cells were incubated for 24 h with testosterone substrate (200 ng-ml') in the presence or not (control) of FSH (100 ng-ml~') and of bFGF increasing concen- trations Determination of estradiol production was performed by radioimmunoassay Values are expressed in pg per 10° cells and are representative of three experiments (mean + SEM) **, Significantly different at P< 0.01; ***, significantly different at P < 0.001 from FSH values NS, not significant.
Trang 4Table 2 bFGF effect on cAMP-stimulated estradiol synthesis in
immature cultured rat Sertoli cells Sertoli cells were incubated for 24 h
with testosterone substrate (200 ng-mL~') (control) in the presence of
1mm dbcAMP or 10 pgmL” cholera toxin and/or 5 ngmL of
bFGF Estradiol production was determined by radioimmunoassay
Values are expressed in pg per 10° cells and are representative of three
experiments (mean + SEM)
Estradiol (pg per 10° cells)
dbcAMP (1 mm) + bFGF (5 ng mL’) 244 + 16
Cholera toxin (10 pg:mL~') 379 + 29
Cholera toxin (10 ugmL~”) + bFGF 227 + 18
(5 ng-mL')
decrease of FSH-stimulated estradiol synthesis by stimulat-
ing cAMP-specific 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 ngmL7' 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 with 100 ngmL~' FSH and
5ngmL ` bFGE In these conditions, cytochrome P450
aromatase mRNA expression was inhibited (—41%) by
comparison to FSH taken as control (Fig 3) A similar
bFGF inhibitory effect on P450 aromatase mRNA expres-
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 modify inhibitory effect of exogenous bFGF on FSH-
stimulated estradiol synthesis Indeed, Sertoli cells are
bFGF producing cells [2,33] and are the target of this
growth factor as bFGF inhibits FSH-induced estradiol
synthesis ({5], and our results) HSPG and especially
glypicans and syndecans 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 sulfate donor for sulfotrans-
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 10 mm sodium
chlorate for 24 h, an increase of FSH-stimulated estradiol
production (+ 42.5%) was observed as described previously
[38] Addition of 10 mm NaCl, used as negative control,
did not induce any modification of FSH-stimulated estradiol
synthesis (data not shown) However, concomitant treat-
800 -
400 -
0 FSH FSH FSH (100ng/ml) +Ro-20-1724 +Ro-20-1724
(Sng/ml) Fig 2 Inhibitory effect of bFGF on 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 24h with testosterone substrate (200 ng-‘ml_'), FSH (100 ng-ml7') and/or Ro-20-
1724 (250 uM) and/or bFGF (5 ngml”') Estradiol production was determined by radioimmunoassay Values are expressed in pg per 10° cells and are representative of three experiments (mean + SEM)
* significantly different at p < 0.05 from FSH or FSH + Ro-20-1724 values
ment of bFGF with 10 mm sodium chlorate totally abolished the inhibitory bFGF effect previously observed on Sertoli cell estradiol synthesis (Fig 4) Similar results were obtained when Sertoli cells were incubated with 1mm dbcAMP instead of FSH (data not shown) Thus, abolition by sodium chlorate of bFGF effect on FSH-stimulated steroidogenesis could implicate HSPG in the bFGF signaling
bFGF effect on cell attachment
Addiion of 5ngmL ' bFGF did not promote any significant difference in cell attachment to substratum (data not shown) as the DNA content of the cell layer at the end
of the 24 h incubation period was identical 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, biochemical and functional modifications as previously mentioned Thus, the relationship between bFGF and HSPG 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 24h with
100 ngmL~' FSH, estradiol production decreased with developing Sertoli cells (Table 3)
When Sertoli cells from 10-day-old-rats were incubated for 24h with 100 ngmL”’ FSH and 5 ngmL! bFGF, FSH-stimulated estradiol production decreased (—40%) This inhibition was less important than in Sertoli cells from 20- and 30-day-old rats (-49 and —53%, respectively) (Table 3) Thus, bFGF inhibitory effect was more pro- nounced on FSH-stimulated estradiol production with
Trang 5
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(Ing/ml) (Sng/mÌ) (10ng/ml)
Fig 3 Dose-related effect of bFGF on FSH-stimulated P450 aromatase mRNA in immature cultured rat Sertoli cells Sertoli cells were incubated for
24 h in the absence of 100 ng-ml~' FSH (lane 1) or in the presence of 100 ng-mI~! FSH and increasing concentrations of bFGF (lanes 2 to 6) Total RNA was extracted as described in Materials and methods Then, 500 ng RNA was reverse-transcribed and amplified by relative quantitative RT-PCR as previously described (A) Agarose gel of one representative experiment (Bl) The densitometry data are representative of three experiments (mean + SEM) Aromatase mRNA level under treatment is expressed versus control which is arbitrarily set to 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%
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 ngmL~' FSH, 10 mm sodium chlorate and 5 ngmL7! bFGF for 24 h, bFGF inhibitory effect on
Trang 6
Fig 4 Effect of bFGF in the presence of sodium chlorate on FSH-
stimulated estradiol synthesis in immature rat Sertoli cells Sertoli cells
were incubated with testosterone substrate (200 ng-ml_') (control) in
the presence of FSH (100 ng-mL') and/or bFGF (5 ngmL*') and/or
sodium chlorate (10 mm) during 24h Estradiol production was
determined by radioimmunoassay Values are expressed in pg per 10°
cells and are representative of three experiments (mean + SEM)
** Significantly different at P < 0.01 from FSH values NS, not
significant
FSH-stimulated estradiol synthesis was not observed
(Table 3) In conclusion, bFGF requires cell surface HSPG
for the inhibition of steroidogenesis in developing Sertoli
cells
Cell surface HSPG are bFGF targets during Sertoli cell
postnatal development
As previously mentioned, glypican-l, syndecan-! and
syndecan-4 are coreceptors for bFGF in some cellular
models As bFGF requires cell surface HSPG to regulate
FSH-stimulated estradiol production, we evaluated if bFGF
itself could influence syndecan-1, syndecan-4 and glypican-1
mRNAs expression
bFGF effect on glypican-1, syndecan-1 and syndecan-4
mRNAs expression
The relative mRNA expression of these HSPG was
evaluated using semi-quantitative RT-PCR as described
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
significantly increased between 10- and 20-days old, then
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- and 20-days old, then decreased between 20- and 30-days-old but however, was 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 ngmL~' bFGF, bFGF inhibited glypican-1 mRNA expression (—35%) but had no effect 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 ngmL7! bFGF, glypican-1 mRNA expression was inhibited (—37%) similarly to 10-day-old cells whereas syndecan-l and syndecan-4 mRNAs expression was not modified compared to the 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 first time in developing rat Sertoli cells that HSPGs are partners for bFGF signal transduction
as coreceptors, and 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 decreased cytochrome P450 aromatase mRNA expression 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 level and could also, 1n 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 testis development [40,41] In this way, the inhibitory 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 of Sertoli cells to estrogens impairs or delays their functional maturation
Table 3 bFGF effect on FSH-stimulated estradiol synthesis in the presence or not of cell surface HSPG during Sertoli cell development Sertoli cells were incubated for 24 h with testosterone substrate (200 ng-mL~') in the presence or not (control) of FSH (100 ng-‘mL7') and/or bFGF (5 ng-mL~') and/or sodium chlorate (10 mm) Estradiol production was determined by radioimmunoassay Values are expressed in pg per 10° cells and are representative of three experiments (mean + SEM) for each studied age
Estradiol synthesis (pg per 10° cells)
Sertoli cells
Trang 7
A 10 days 20 days 30 days
2500
2000 -
4500 -
1000
Glypican-1 Syndecan-1
The bFGF binding requires specific 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 developing Sertoli
cells Thus, inhibitory effect of bFGF on FSH-stimulated
estradiol production requires the 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-l, syndecan-l and
syndecan-4 are potential coreceptors of bFGF We indicated
that the HSPG mRNA pattern is not similar in Sertoli cells
from 10, 20 and 30 days old In addition, glypican-1 mRNA
expression was inhibited (-35% and —37%, respectively) in
Sertoli cells from 10- and 20-day-old rats as described in
oligodendrocytes [43] or in lung fibroblasts [44] In contrast,
this inhibition was abolished in 30-day-old rat Sertoli cells
Syndecan-1 mRNA expression was not modified 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-old rats Thus, bFGF effect seems
to be HSPG-, developmental stage- and cell type-specific
Indeed, bFGF does not regulate syndecan-1 mRNA
expression in MCA3D keratinocytes [45,46] or in endothelial
cells [47] However, this growth factor increases syndecan-1
and syndecan-4 mRNAs expression in fibroblasts [48] and in
vascular smooth muscle cells [49], respectively
At the transcriptional level, the 5’ flanking region of
syndecan-l contains an FGF-inducible response element
#E,Ì
Syndecan-4
Glyp-l Synd-l Synd-4
B-Actin
Fig 5 Evolution of glypican-1, syndecan-1 and syndecan-4 mRNAs expression during Sertoli cells development Sertoli cells from 10-, 20- and 30-day-old rats were incubated for 24 h without treatment 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 [22] 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 experiments (mean + SE) for each age
6 -Actin
(FiRE) [50] In this study, bFGF increases syndecan-1 mRNA expression in Sertoli cells from 30-day-old rats This observation suggests that these cells might express all transcription factors components of FiRE, namely USF, the uncharacterized p46 nuclear proteins, AP-1 (Jun/Fos) complexes and a putatively novel FGF-inducible nuclear protein-1 (FIN-1) [50] These transcriptional elements are differentially regulated depending on cell type and activating growth factor [50] In NIH 3T3 fibroblasts, FiRE was shown to be selectively induced by bFGF whereas in keratinocytes, FiRE was not induced by this growth factor Whether FiRE is really expressed in Sertoli cells or not, some post-translational modifications, phosphorylation or dephosphorylation of FiRE components could contribute
to the specificity [51-54] Inhibitory transcription factors that bind to AP-1 or FIN-1 or that inactive binding 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 regulates differently HSPG expression upon Sertoli cell maturation suggesting a func- tional selectivity
Assuming that levels of glypican-1 and syndecan protein
synthesis correlate well with mRNAs levels, it is likely that,
in the presence of bFGF, the plasma membrane will be enriched with syndecans and a decrease of glypican-1 Further experiments will be needed to understand the biological significance of the different regulation of their expression As a first element step towards 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
Trang 8
A
10 Days
Glyp-1 Synd-l ynd-4 bFGF bFGF
(10 ng/ml) ~ 3 (10 ng/ml)
=
2500 - im 20 days
5 (130 days
>
2 — 2000 -
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a+
$ 5 1000
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10 ng/ml
+
30 Days
Glyp-l ynd-l Synd-4
bFGF
Fig 6 Action of bFGF on Glypican-1, syndecan-1 and syndecan-4 mRNAs expression during Sertoli cells development Sertoli cells from 10-, 20- and 30-day-old rats were incubated for 24 h in the presence (+) or in the absence (-) of 10 ngml~! bFGF 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 [22] Glyp-1, glypican-1; Synd-1, syndecan-1; Synd-4, syndecan-4 (A) Agarose gel of one representative experiment for each studied age (B) Densi- tometry data are representative of three, five and three different experiments (mean + SE) for 10-, 20- and 30-day-old-rat Sertoli cells, respectively
HSPG in the decrease of estradiol production [55] The
aromatase activity decreases by modulation of cytoskeleton
occuring during Sertoli cell development [56] In this way,
syndecan-| and syndecan-4 could participate to this event in
reorganizing actin filaments via their cytoplasmic domain
[57] but also in the presentation and delivery of bFGF to its
receptors Moreover, during Sertoli cell development,
phosphodiesterase activity increases [58] Phamanthu ef al
[38] suggest a possible involvement of cell HSPG in the age-
related increase in Sertoli cell phosphodiesterase activity and
in the concomitent loss of steroidogenic response to FSH
These data and our results suggest that bFGF could
modulate, in part, the decrease of FSH-stimulated estradiol
synthesis via HSPG
REFERENCES
1 Nishimura, T., Nakatake, Y., Konishi, M & Itoh, N (2000)
Identification of a novel FGF, FGF-21, preferentially expressed in
the liver Biochim Biophys Acta 1492, 203-206
2 Han, IS., Sylvester, S.R., Kim, K.H., Schelling, M.E., Venkateswaran, S., Blanckaert, V.D., McGuinness, M.P & Griswold, M.D (1993) Basic fibroblast growth factor is a testi- cular germ cell product which may regulate Sertoli cell function Mol Endocrinol 7, 889-897
Bikfalvi, A., Klein, S., Pintucci, G & Rifkin, D.B (1997) Biological roles of fibroblast growth factor-2 Endrocrin Rev 1,
26-45
Laslett, A.L., McFarlane, J.R., Hearn, M.T.W & Risbridger, G.P (1995) Requirement for heparan sulphate proteoglycans to mediate basic fibroblast growth factor (FGF-2) -induced stimu- lation of Leydig cell steroidogenesis J Steroid Biochem Molec
Biol 54, 245-250
Schteingart, H.F., Meroni, $.B., Capena, D.F., Pellizzari, EH & Cigorraga, S.B (1999) Effects of basic fibroblast growth factor and nerve growth factor on lactate production, y-glutamyl trans- peptidase and aromatase activities in cultured Sertoli cells Eur J Endocrinol 141, 539-545
Boockfor, F.R & Schwarz, L.K (1990) Fibroblast growth factor modulates the release of transferrin from cultured Sertoli cells Mol Cell Endocrinol 73, 187-194.
Trang 97
10
IL
12
13
14
15
16
17
18
19
20
21
22
23
24
Jaillard, C., Chatelain, P.G & Saez, J.M (1987) In vitro regula-
tion of pig Sertoli cell growth and function: effects of fibroblast
growth factor and somatomedin-C Biol Reprod 37, 665-674
Smith, E.P., Hall, S.H., Monaco, L., French, $.H., Wilson, E.M
& Conti, M (1989) A rat Sertoli cell factor similar to basic fi-
broblast growth factor increases c-fos messenger ribonucleic acid
in cultured Sertoli cells Mol Endocrinol 3, 954-961
Le Magueresse-Battistoni, B., Wolff, J Morera, A.M &
Benahmed, M (1994) Fibroblast growth factor receptor type 1
expression during rat testicular development and its regulation in
cultured Sertoli cells Endocrinology 135, 2404-2411
Nugent, M.A & Iozzo, R.V (2000) Fibroblast growth factor-2
Int J Biochem Cell Biol 32, 115-120
Yayon, A., Klagsbrun, M., Esko, J.D., Leder, P & Ornitz, D.M
(1991) Cell surface, heparin-like molecules are required for binding
of basic fibroblast growth factor to its high affinity receptor Cell
64, 841-848
Rapraeger, A.C., Krufka, A & Olwin, B.B (1991) Requirement of
heparan sulfate for bFGF-mediated fibroblast growth and myo-
blast differenciation Science 252, 1705-1708
Heath, W.F., Cantrell, A.S., Mayne, H.G & Richard, J.S (1991)
Mutations in the heparin binding domains of human basic fibro-
blast growth factor alter its biological activity Biochemistry 30,
5608-5615
Savona, C., Chambaz, E.M & Feige, J.J (1991) Proteoheparan
sulfate contribute to the binding of basic fibroblast growth factor
to its high affinity receptors on bovine adrenocortical cells Growth
Factor 5, 273-282
Ornitz, D.M., Yayon, A., Flanagan, J.G., Svahn, C.M., Levi, E &
Leder, P (1992) Heparin is required for cell-free binding of basic
fibroblast growth factor to a soluble receptor and for mitogenesis
in whole cells Mol Cell Biol 12, 240-247
Dorrington, J.H & Fritz, I1.B (1975) Androgen synthesis and
metabolism by preparations from the seminiferous tubule of the
rat testis In Hormonal Regulation of Spermatogenesis (French,
F.S., Hansson, V., Ritzen, E.M & Nayfeh, S.N., eds.), pp 37-52
Plenum Press, New York
Welsh, M.J & Wiebe, J.P (1976) Sertoli cells from immature rats:
in vitro stimulation of steroid metabolism Biochem Biophys Res
Commun 69, 936-941
Suarez-Quian, C.A., Dym, M., Makris, A., Brumbaugh, J., Ryan,
K.J & Canick, J.A (1983) Estrogen synthesis by immature rat
Sertoli cells in vitro J Androl 4, 203-209
Papadopoulos, V., Carreau, S., Szerman, J.E., Drosdowsky,
M.A., Dehennin, L & Scholler, R (1986) Rat testis 176-estradiol:
identification by gas chromatography-mass spectrometry and
age related cellular distribution J Steroid Biochem 24, 1211-
1216
Mounis, A., Barbey, P., Langris, M & Bocquet, J (1991) Deter-
gent-solubilized proteoglycans in rat testicular Sertoli cells Bio-
chim Biophys Acta 1074, 424-432
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
Brucato, S., Harduin-Lepers, A., Godard, F., Bocquet, J &
Villers, C (2000) Expression of glypican-1, syndecan-1 and
syndecan-4 mRNAs protein kinase C-regulated in rat immature
Sertoli cells by semi-quantitative RT-PCR analysis Biochim
Biophys Acta 1474, 31-40
Filla, M.S., Dam, P & Jalkanen, M (1998) The cell surface
proteoglycan syndecan-1 mediates fibroblast growth factor-2
binding and activity J Cell Physiol 147, 310-321
Steinfeld, R., Van den Berghe, H & David, G (1996) Stimulation
of fibroblast growth factor receptor-1 occupancy and signaling by
cell surface-associated syndecans and glypican J Cell Biol 133,
405-416
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
4I
42
Bonneh-Barkay, D., Shlissel, M., Berman, B., Shaoul, E., Admon, A., Vlodavsky, I., Carey, D.J., Asundi, V.K., Reich Slotky, R & Ron, D (1997) Identification of glypican as a modulator of the activity of fibroblast growth factors J Biol Chem 272, 12415-
12421
Carey, D.J (1997) Syndecans:
co-receptors Biochem J 327, 1-16
Tung, P.S., Skinner, M.K & Fritz, IB (1984) Fibronectin syn- thesis is a marker for peritubular contaminants in Sertoli cell- enriched cultures Biol Reprod 30, 199-211
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
Chomezynski, P & Sacchi, N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction Anal Biochem 162, 156-159
Carreau, S & Levallet, J (1997) Cytochrome P450 aromatase in male germ cells Folia Histochem Cytobiol 35, 195-202 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
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
Mullaney, B.P & Skinner, M.K (1992) Basic fibroblast growth factor (bFGF) gene expression and protein production during pubertal development of the seminiferous tubule: follicle-stimu- lating hormone-induced Sertoli cell bFGF expression Endocri- nology 131, 2928-2934
Aviezer, D., Levy, E., Safran, M., Svahn, C., Buddecke, E., Schmidt, A., David, G., Vlodavsky, I & Yayon, A (1994) Dif- ferential structural requirements of heparin and heparan sulfate proteoglycans that promote binding of basic fibroblast growth factor to its receptor J Biol Chem 269, 114-121
Brauer, P.R., Keller, K.M & Keller, J.-M (1990) Concurrent reduction in the sulfation of heparan sulfate and basement membrane assembly in a cell model system Development 110, 805-
813
Greeve, H., Cully, Z., Blumberg, P & Kresse, H (1988) Influence
of chlorate on proteoglycan biosynthesis by cultured human
fibroblasts J Biol Chem 263, 12888-12891
Humphries, D.E & Silbert, J.E (1988) Chlorate: a reversible inhibitor of proteoglycan sulfation Biochem Biophys Res Comm
145, 365-371
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 production in immature rat Sertoli cells J Androl 20,
241-250
Morera, A.M., Esposito, G., Ghiglieri, C., Chauvin, M.A., Hartmann, D.J & Benhamed, M (1992) Transforming growth factor B1 inhibits gonadotropin action in cultured porcine Sertoli cells Endocrinol 130, 831-836
Ackland, J.F., Schwartz, N.B., Mayo, K.E & Dodson, R.E (1992) Nonsteroidal signals originating in the gonads Physiol Rey 72, 731-765
Rosselli, M & Skinner, M.K (1992) Developmental regulation of Sertoli cell aromatase activity and plasminogen activator pro- duction by hormones, retinoids and the testicular paracrine factor, PModS Biol Reprod 46, 586-594
Sharpe, R.M., Atanassova, N., McKinnell, C., Parte, P., Turner, K.J., Fisher, J.S., Kerr, J.B., Groome, N.P., Macpherson, S., Millar, M.R & Saunders, P.T (1998) Abnormalities in functional development of the Sertoli cells in rat treated neonatally with diethylstillbestrol: a possible role for estrogens in Sertoli cells development Biol Reprod 59, 1084-1094
multifonctional cell-surface
Trang 104
A4
45
46
47
48
49
50
Bansal, R., Kumar, M., Murray, K & Pfeiffer, S.E (1996) De-
velopmental and FGF-2-mediated regulation of syndecans (1-4)
and glypican in oligodendrocytes Mol Cell Neurosc 7, 276-288
Romaris, M., Bassols, A & David, G (1995) Effect of trans-
forming growth factor-bl and basic fibroblast growth factor on
the expression of cell surface proteoglycans in human lung fibro-
blasts Biochem J 310, 73-81
Tsuboi, T., Sato, C., Kurita, Y., Ron, D., Rubin, J.S & Ogawa,
H (1993) Keratinocyte growth factor (FGF-7) stimulates migra-
tion and plasminogen activator activity of normal human kerati-
nocytes J Invest Dermatol 101, 49-53
Jaakkola, P., Maatta, A & Jalkanen, M (1998) The activation
and composition of FiRE (an FGF-inducible response element)
differ in a cell type- and growth factor-specific manner Oncogene
17, 1279-1286
Kainulainen, V., Nelimarkka, L., Jarvelainen, H., Laato, M.,
Jalkanen, M & Elenius, K (1996) Suppression of syndecan-1
expression in endothelial cells by tumor necrosis factor-alpha
J Biol Chem 271, 18759-18766
Jaakkola, P., Vihinen, T., Maatta, A & Jalkanen, M (1997)
Activation of an enhancer on the syndecan-1 gene is restricted to
fibroblast growth factor family members in mesenchymal cells
Mol Cell Bioi 17, 32103219
Cizmeci-Smith, G., Langan, E., Youkey, J., Showalter, L.T &
Carey, D.J (1997) Syndecan-4 is a primary-response gene induced
by basic fibroblast growth factor and arterial injury in vascular
smooth muscle cells Artrioscler Thromb Vasc Biol 17, 172-180
Jaakkola, P & Jalkanen, M (2000) Transcritptional regulation of
syndecan-1 expression by growth factors Prog Nucleic Acid Res
Mol Biol 63, 109-138
51
32
53
54
55
56
57
58
Smeal, T., Binetruy, B., Mercola, D.A., Birrer, M & Karin, M (1991) Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73 Nature
354, 494-496
Baker, S.J., Kerppola, T.K., Luk, D., Vandenberg, M.T., Marshak, D.R., Curran, T & Abate, C (1992) Jun is phospho- rylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts Mol Cell Biol 12,
4694-4705
Franklin, C.C., Sanchez, V., Wagner, F., Woodgett, J.R & Kraft, A.S (1992) Phorbol ester-induced amino-terminal phosphoryl- ation of human JUN but not JUNB regulates transcriptional activation Proc Natl Acad Sci USA 89, 7247-7251
Hill, C.S & Treisman, R (1995) Transcriptional regulation
by extracellular signals: mechanisms and specificity Cel 80,
199-211
Phamantu, N.T., Bonnamy, P.J., Bouakka, M & Bocquet, J (1995) Inhibition of proteoglycan synthesis induces an increase in follicle stimulating hormone (FSH) -stimulated estradiol produc- tion by immature rat sertoli cells Mol Cell Endo 109, 37-4S Meroni, S.B., Steingart, H.F., Pellizzari, E.H & Cigorraga, S.B (1995) Possible involvement of microfilaments in the regulation
of Sertoli cell aromatase activity Mol Cell Endocrinol 112,
69-75
Carey, D.J., Bendt, K.M & Stahl, R.C (1996) The cytoplasmic domain of syndecan-1 is required for cytoskeleton association but not detergent insolubility J Biol Chem 271, 15253-15260 Griswold, M.D (1993) Postnatal Sertoli cell development In The Sertoli Cell (par Russel, L.D & Griswold, M.D., eds), pp 493-
508 Cache River Press, USA.