Regulation of glypican-1, syndecan-1 and syndecan-4 mRNAsexpression by follicle-stimulating hormone, cAMP increase and calcium influx during rat Sertoli cell development Sylvie Brucato,
Trang 1Regulation 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 proteoproteo-glycans (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 cAMPsynthesis and a calcium influx
This study demonstrates that FSH, through its second
messengers (increase in intracellular cAMPand 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 cAMPand 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, cAMPresponse element
binding protein; CREM, cAMPresponse element modulator.
(Received 18 February 2002, revised 8 May 2002,
accepted 29 May 2002)
Trang 2role 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
been identified as both L and N type [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 cAMPphosphodiesterase increase upon ontogenesis
[19]
The present work aims to investigate whether FSH,
intracellular cAMPlevel increase and alteration of
trans-membrane calcium influx induce changes on glypican-1,
syndecans-1 and -4 mRNA expression in developing rat
Sertoli cells
M A T E R I A L S A N D M E T H O D S
Reagents
All reagents were of analytical or molecular biology grade
Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F12
medium, Trypsin (USPGrade), 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 MgCl2 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 cm2 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% CO2in air at 32C Culture medium was renewed after 48 h Three days after plating, residual germinal cells were removed by brief hypotonic treatment using 20 mMTris/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, 55–60C, 5 min) was added
to a reverse transcription reaction mixture containing the reaction buffer (50 mM Tris/HCl, pH 8.3, 50 mM KCl;
10 mMMgCl2, 0.5 mMspermidine, dithiothreitol 10 mM),
1 lM oligo d(T)15, 500 lM dNTPs, 20 U RNasin, 18 U AMV-reverse transcriptase in 20-lL final volume The reaction was carried out at 37C for 60 min, followed by
5 min denaturation at 95C
Two microliters of the first strand synthesis product (0.1 lg) was used as template to amplify each cDNA PCR was performed with 250 lMdNTPs, Taq DNA polymerase reaction buffer (50 mMKCl, 10 mMTris/HCl, pH 9, 0.1% Triton X-100), 2.5 UI Taq DNA polymerase, MgCl2 1.5 mM, 10 pmol of each primer in a 20-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¢
and 5¢-AGCCATGCCAAATGTCTCAT-3¢ for b-actin The PCR was started at 94C for 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
Trang 3previously [24] which consisted of a denaturating step
(at 94C for 1 min), an annealing step (at 55 C for 1 min)
and an extension step (at 72C for 2 min) then a final
elongation step (at 72C for 10 min) in Robocycler
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 (0.04M Tris, acetate, 0.001M EDTA) Gels were
stained with ethidium bromide (1 lgÆmL)1), photographed
using Polaroid film under UV light and analysed using a
AGFA SnapScan 1200
software and the NIHIMAGE 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 1M NaOH of the cell layer and
subsequent neutralization by 1M KH2PO4, DNA was
quantified in a Kontron spectrofluorimeter using Hoescht
33258 as fluorescent probe and calf thymus as standard
R E S U L T S
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 AMPand calcium levels
Effect of intracellular cAMP level increase
The involvement of the cAMPpathway 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 Gsactivator [38]; and
(c) RO-20 1724, a specific cAMPphosphodiesterase
inhib-itor [39]
Twenty-day-old rat Sertoli cells were incubated for 24 h
with increasing concentrations of dbcAMP(0–2 mM) (data
not shown) The mRNA glypican-1 expression inhibition was optimal ()56%) for 1 mMof dbcAMPand maintained for high concentrations of dbcAMP The syndecan-1 mRNA expression was increased by 1 mM 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 dbcAMPeffect was con-firmed by using 10 lgÆmL)1of cholera toxin during 24 h in 20-day-old rat Sertoli cells This agent induced the same effects as the dbcAMPones 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 cAMPphosphodiesterases inhibitor used at 250 lM, 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 cAMPlevel (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).
Trang 4Effect 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 lM 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 mM), 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) and H8 (5 lM), 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 cAMPand 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 cAMPand 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 mM) 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 mM) 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 reprep-resentative 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 (–) of 10 lgÆmL)1cholera 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%.
Trang 5expression 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 dbcAMPupregulated 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 dbcAMPstimulated them by +35 and
+55%, respectively (Tables 2 and 3)
D I S C U S S I O N
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 AMPand
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 cAMPlevel 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 cAMPincrease in 30-day-old rat Sertoli cells
Until now, there has been little information concerning cAMPeffects 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 cAMPlevel in Sertoli cells whereas the total intra-cellular cAMPconcentration was not implied on its increase in NIH 3T3 fibroblasts [41] In vascular smooth muscle (VSM) cells, carbacyclin and forskolin, agents that elevate cAMPlevels, failed to increase syndecan mRNA levels in contrast to our results [42] Moreover, endothelin, which reduces cAMPaccumulation 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 cAMPeffect 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 relarepresenta-tive 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 relarepresenta-tive HSPG
mRNA level under treatment is expressed vs control which is
arbi-trarily set to 100%.
Trang 6The 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 (cAMPresponse element binding protein),
CREM (cAMPresponse 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)
[24] Thus, other PKA- and PKC-inducible elements that remain to be elucided could explain the observed differential regulation
Beyond increased cAMPsynthesis, 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 syndeglypi-can-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) and H8
(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 relarepresenta-tive 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)1FSH,
1 mM dbcAMP(intracellular cAMPincrease), or 100 ngÆmL)1FSH
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
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 cAMPincrease), 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
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 cAMPincrease), 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
Trang 7Thus, FSH, as the increase in intracellular cAMPand
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 cAMPand 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 cAMPbut 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
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