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INSERM, U-407, Communications Cellulaires en Biologie de la Reproduction, Faculté de Médecine Lyon-Sud (I.G, M.B., M.B.), 69921 Oullins, France; INSERM, U-244, CEA Grenoble (M.K., J.J.F.), 38054 Grenoble, France; and Laboratoire de Radioimmunologie, Université de Liège (J.C.H.), 4000 Liège, Belgium
Address all correspondence and requests for reprints to: Dr. M. Benahmed, INSERM, U-407, Communications Cellulaires en Biologie de la Reproduction, Faculté de Médecine Lyon-Sud, BP 12, F-69921 Oullins Cedex, France. E-mail: benahmed{at}lsgrisn1.univ-lyon1.fr
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, transforming
growth factor-ßs (TGFßs), cytokines (interleukin-1 and -6, tumor
necrosis factor-, and stem cell factor), and neurotropins (nerve
growth factor and brain-derived growth factor). In mammals, the TGFß
peptide family comprises closely related peptides, including TGFß1,
-2, and -3, that share high homology between species and more distant
peptides, such as inhibins, activins, anti-Mullerian hormone (AMH), and
bone morphogenetic proteins (BMPs) (4, 5). The critical role of the TGFß peptide family in male gonadal development and function is supported by at least three types of observations. Firstly, TGFß family peptides have been shown to be present in the testis. Secondly, TGFß1 has been largely shown to modulate different testicular activities mainly in in vitro systems. For example, TGFß1 has been reported to be a modulator of hormone formation in both cultured Leydig and Sertoli cells and of contractility, shape, and organization of peritubular myoid cells (for reviews, see Refs. 2, 3). Some TGFß-related peptides have also been reported to affect testicular functions and particularly Leydig cell steroidogenesis. Among these factors are inhibins and activins (6, 7) and AMH (8). Thirdly, recent observations in transgenic models in which genes related to the TGFß peptide families were manipulated (knockout, overexpression) indicated that the reproductive function in these animals was affected. For example, in TGFß2-deficient mice, testis hypoplasia, cryptorchidism, and ectoplasia were observed (9); in BMP8-deficient mice, the germ cells of all homozygote mutants either failed to proliferate or showed a marked reduction in proliferation and a delayed differentiation (10). Overproduction of TGFß1 may also affect the testis with atrophy of the gonad and thickened tubular basement membranes (11). Together, these data clearly indicate that the action of the TGFß peptide family is probably of major importance for the testis in terms of both development and function (i.e. steroidogenesis and gametogenesis).
However, paradoxically, although the testicular activity of the ligands TGFß and related peptides have been largely studied, very little is known about their testicular receptors, obligatory intermediates in their action. With exception of immunohistochemical approaches to localization (12, 13), such receptors have not yet been characterized in terms of proteins and messenger RNAs (mRNAs) in the testis. It is known that the members of the TGFß superfamily transduce signals through two different types of serine/threonine protein kinase receptors, known as type I and type II receptors (5). TGFß superfamily ligands bind to the TGFß type II receptor (TGFßRII), which has a constituitively active kinase; the TGFß type I receptor (TGFßRI) is then recruited into the TGFß/TGFßRII complex, and this results in the activation of TGFßRI kinase (14). The TGFßRI kinase transduces intracellular signals by activation of various proteins, including Smad proteins. The signal is transferred to the Smad proteins through the receptor kinase-mediated phosphorylation of pathway-specific Smads. The signal is then propagated primarily through protein-protein interactions between Smad proteins, which are heterooligomeric, and between Smads and transcription factors. Specifically, Smads are the mediators not only for TGFß, but also for activins, AMH and BMPs. Specifically, Smads 1, 5, and 8 appear as substrates for the BMP type I receptor kinase and the orphan receptor ALK1. Smads 2 and 3 are phosphorylated by activated activin or TGFßRI. Smad 4 is a common mediator, as it forms heterooligomeric complexes with other activated Smads. The inhibitory Smads (anti-Smads, Smads 6 and 7) antagonize the activity of the Smad 4-and other Smads (i.e. 1, 2, 3, 5, 8) complexes (for reviews, see Refs. 5, 15).
By using a model of purified cultured Leydig cells, the aim of the present study was to 1) identify and characterize TGFß receptors and Smads in this testicular cell type and 2) determine whether the expression of the TGFß receptors is under the control of the endocrine system exerted through LH/hCG action on Leydig cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]deoxy (d)-CTP were obtained from
Amersham Pharmacia Biotech (Aylesbury, UK).
Oligonucleotide primers were obtained from Genset (Paris, France).
Molonry murine leukemia virus was obtained from Life Technologies, Inc. (Eragny, France), and Taq
polymerase was purchased from Promega Corp. (Lyon,
France). Human TGFß type I receptor (ALK-5) complementary DNAs (cDNAs) (16), and porcine TGFß type III receptor cDNAs (17) were provided by Dr. K. Miyazono (Uppsala, Sweden). Porcine TGFß type II receptor cDNAs (18) was provided by Dr. X. F. Wang (Durham, NC). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were a gift from Dr. J. M. Blanchard (Faculté des Sciences, Montpellier, France).
Leydig cell preparation and culture
Isolated Leydig cells were prepared from porcine testes (2–3
weeks old) by collagenase treatment as described by Mather and Phillips
(19) and modified by Benahmed et al. (20). Briefly,
decapsulated testes were minced and washed in DMEM/Ham’s F-12 medium
(1:1). After collagenase dissociation (0.5 mg/ml, 90 min at 32 C),
cells were washed by centrifugation (200 x g for 10
min). The pellets were then resuspended and submitted to two successive
sedimentations of 5 and 15 min. The crude interstitial cells were
recovered from the supernatants, and Leydig cells were prepared from
this fraction by Percoll gradient centrifugation. The purity of Leydig
cells was more than 90%, as determined by histochemical
3ß-hydroxysteroid dehydrogenase staining.
Leydig cells were plated in Falcon (Los Angeles, CA) six-multiwell plates (2 x 106 cells/dish) or 100 x 20-mm petri dishes (107 cells/dish) and cultured at 32 C in a humidified atmosphere of 5% CO2-95% air in DMEM/Ham’s F-12 medium (1:1) containing sodium bicarbonate (1.2 mg/ml), 15 mM HEPES, gentamicin (20 µg/ml), and nystatin (20 IU/ml). This medium was supplemented with insulin (2 µg/ml), transferrin (5 µg/ml), and vitamin E (10 µg/ml).
Testosterone production
Basal and LH/hCG-stimulated testosterone secretion was measured
in the culture medium using specific RIA (21).
125I labeling of TGFß1
TGFß1 was purified according to the method described by Cone
et al. (22). Purified TGFß1 (1 µg) was labeled with
125I by the chloramine-T method following the
procedure described by Frolik et al. (23). Briefly, 1 µg
TGFß1 was diluted with 10 µl 1.5 M potassium
phosphate, pH 7.4, and 10 µl [125I]Na. To
initiate the reaction, a 5-µl aliquot of chloramine-T solution (0.1
mg/ml) was added. After 2 min at room temperature, an additional 5-µl
aliquot was added, followed 1.5 min later by final a 5-µl aliquot.
One minute after the last addition of chloramine-T, 25 µl 50
mM N-acetyltyrosine were added. After
2 min, 200 µl 60 mM potassium iodide and 200
µl 8 M urea were added. The mixture was run
through an equilibrated PD-10 Pharmacia Biotech column and
eluted with 4 mM HCl, 75 mM
sodium chloride, and 0.1% BSA. Preparations of
[125I]TGFß1 with a specific activity of about
5–10 x 106 cpm/pmol, determined by
trichloroacetic acid precipitation, were obtained by this method.
Affinity cross-linking of TGFß receptors
Leydig cell monolayers (cultured in six-multiwell plates) were
affinity labeled with [125I]TGFß1 as
described by Goddard et al. (24). Briefly, cultured Leydig
cells, previously incubated with [125I]TGFß1,
were treated with 0.3 mM disuccinimidyl suberic
acid (Pierce Chemical Co., Rockford, IL) for 15 min at 0
C. The cells were then washed and scraped off in the presence of
ice-cold 10 mM Tris buffer (pH 7.0) containing
0.25 M sucrose, 1 mM EDTA,
1 mM phenylmethylsulfonylfluoride, and 10 µg/ml
leupeptin. After centrifugation of the extracts, the supernatants were
adjusted to a final concentration of 10% glycerol, 2% SDS, and 1.3%
2ß-mercaptoethanol before SDS-PAGE analysis. SDS-PAGE was performed
according to the Laemmli procedure (25), using 6% polyacrylamide flat
gels. At the end of the separation, the gels were stained for proteins
with Coomassie blue, dried, and then autoradiographed using
Amersham Pharmacia Biotech Hyperfilm-MP.
14C-Methylated molecular mass markers were myosin
(200 kDa), phosphorylase b (97.4 kDa), and BSA (69 kDa; Rainbow
14C-methylated protein molecular mass markers
Amersham Pharmacia Biotech).
It is assumed that each of the affinity-labeled complexes contains a monomer of TGFß (electrophoresis was conducted under reducing conditions that dissociate disulfide-linked TGFß dimers). Throughout this study, the molecular mass of the TGFß monomer (12 kDa) was subtracted to the estimated size of the TGFß-binding protein.
Western blotting analysis
Leydig cells cultured in 100 x 20-mm petri dishes
(107 cells/dish) were incubated in 1 ml RIPA
buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8) with freshly
added phenylmethylsulfonylfluoride (10 µg/ml) for 1 h on ice.
Cells were then scraped and centrifuged at 15,000 x g
for 20 min at 4 C. Proteins from Leydig cells were resolved on 7.5%
SDS-PAGE gels and electrophoretically transferred to nitrocellulose
membranes using 25 mM Tris and 185
mM glycine, pH 8.3, containing 20% methanol. The
transfer was performed at a constant voltage of 100 V for 1 h. The
membranes were blocked for 1–2 h in Tris-buffered saline with Tween 20
[TBST; 10 mM Tris-HCl (pH 8.0), 150
mM NaCl, and 0.05% Tween 20] containing 5%
milk (TBSTM) and rinsed three times in TBST. The nitrocellulose
membranes were incubated with 500 ng/ml (1:200 dilution) of the
polyclonal anti-TGFß receptor type I or type II (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in TBSTM overnight at 4 C.
The membranes were rinsed three times in TBST and incubated for 1
h at room temperature in TBSTM containing antirabbit biotinylated
antibodies (from donkey; 1:400 dilution). The membranes were then
rinsed three times and incubated with streptavidin-biotinylated
horseradish peroxidase complex (1:3000 dilution) for 30 min at 20 C.
After washing the membranes were colored in a solution of
diaminobenzidene chromogen (10 mg in 17 ml TBS with 17 µl
H2O2, 30%). Molecular mass
markers were myosin (207 kDa), ß-galactosidase (139 kDa), BSA (84
kDa), and carbonic anhydrase (41 kDa; Kaleidoscope prestained
standards, Bio-Rad Laboratories, Inc., Richmond, CA).
Northern blotting analysis
Total RNAs were extracted from Leydig cells cultured in petri
dishes, as described by Chomczynski and Sacchi (26). Briefly, cells
were scrapped in 4 M guanidine thiocyanate, 25
mM trisodium citrate, 0.5% sarcosyl, and 0.1 M
2ß-mercaptoethanol, followed by a phenol-chloroform extraction in the
presence of 0.2 M sodium acetate, pH 4. After precipitation
with isopropanol, RNAs were washed with 75% ethanol. After
solubilization with water, RNA quantities were estimated by
spectrophotometry at 260 nm.
Forty micrograms of total RNAs (denatured for 15 min at 65 C in the
presence of 2.2 M formaldehyde and 12.5 M
formamide) were loaded on 1.2% agarose-2.2 M formaldehyde
gels for electrophoretic separation, then transferred to nitrocellulose
membranes (Hybond-C extra, Amersham Pharmacia Biotech) by
capillary transfer with 10 x SSC (1.5 M NaCl and 0.15
M sodium citrate) and fixed at 80 C for 2 h. cDNA
probes (ALK-5, porcine TR-II, or porcine TR-III) were labeled with
[-32P]dCTP using a random primer labeling
kit (Promega Corp.). Labeled probes were separated from
free nucleotides by filtration through diethylaminoethyl-cellulose
column.
After 5–6 h of prehybridization at 42 C, filters were hybridized overnight with labeled probes (2 x 106 cpm/ml ALK-5, porcine TR-II or porcine TR-III) at 42 C in 50% formamide, 5 x SSPE (0.9 M NaCl, 0.05 M sodium phosphate, and 5 mM EDTA, pH 7.4), 5 x Denhardt’s solution (1 g Ficoll, 1 g polyvinyl pyrrolidone, and 1 g BSA/liter), 0.1% SDS, 10% dextran sulfate, and 100 µg/ml transfer RNA from baker’s yeast. The filters were then washed four times in 2 x SSC-0.1% SDS at room temperature for 20 min and twice in 0.1 x SSC-0.1% SDS at 55 C for 20 min. The filters were autoradiographed using Amersham Pharmacia Biotech Hyperfilm-MP.
RT-PCR analysis
Single stranded complementary DNAs (cDNAs) were obtained from RT
of 3 µg total RNAs using random hexanucleotides as primers (5
µM) in the presence of dNTPs (25 µM),
dithiothreitol (10 µM), and Moloney murine leukemia virus
(10 U/µl) for 1 h at 37 C. cDNAs (1 µl of RT mixture) were
amplified by PCR with Taq polymerase (0.04 U/µl), dNTPs
(20 µM), and specific primers (0.4
µM). The mixture was first heated at 95 C for 5
min, and then 35 cycles of 95 C for 40 sec, 55 C for 1 min, 72 C for 2
min, and 72 C for 10 min. PCR products were analyzed on 2% agarose
gels and visualized using a UV (254 nm) table. The specific
oligonucleotide primers, designed to amplify sequences of the different
Smad cDNAs, are shown in Table 1.
Data analysis
The band densities were determined by densitometric analysis
using a Bioimage scanner (Bio Image UK, Cheshire, UK).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the autoradiogram of an
electrophoretic analysis of Leydig cell membranes cross-linked with
[125I]TGFß1 in the absence or presence of
unlabeled TGFß1. Three major labeled cross-linking species of about
300, 80, and 53 kDa were present in Leydig cells (Fig. 1A). These
TGFß-binding proteins correspond to the TGFßRIII, -RII, and -RI,
respectively.
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By using Western blotting analysis with specific polyclonal antibodies,
the presence of both TGFßRI and TGFßRII was confirmed, with their
molecular masses were about 60 and 90 kDa, respectively (Fig. 1B).
TGFßRI, -RII, and -RIII were also detected in forms of mRNA in the
Leydig cells. As shown in Fig. 1, C-I, a single TGFßRI transcript of
5.8 kb was observed. A single TGFßRII transcript was identified at
4.6 kb (Fig. 1, C-II). A single transcript for TGFßRIII of 6.4 kb was
detected (Fig. 1, C-III) in cultured Leydig cells.
Identification of Smads, the mediators of the TGFß signal
Using a RT-PCR approach, it is shown in Fig. 2 that cultured Leydig cells express the
specific mRNAs for Smads 1–7.
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Addition of hCG (from 0.1–10 ng/ml) to Leydig cells resulted, after
48 h of treatment, in a dose-dependent increase in both the 53-
and 80-kDa bands/signals corresponding to TGFßRI and -RII,
respectively. The maximal stimulatory effect was obtained with 0.3
ng/ml hCG (Fig. 3A). As shown in Fig. 3B, this stimulatory effect of hCG was time dependent. After 48 h of
hCG treatment, TGFßRI and -RII levels increased about 12- and
3.5-fold, respectively.
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and 5 show that in hCG-treated Leydig cells,
the stimulation of TGFßRI and -RII proteins was related to an
increase in the level of their corresponding specific mRNAs. For
TGFßRI mRNA, Fig. 4A indicates that the maximal effect (2-fold
increase) was obtained with 2.5 IU/ml hCG. For TGFßRII mRNA, Fig. 5A
indicates that the maximal effect (2-fold increase) was obtained with
0.61 IU/ml hCG. The stimulatory effect of gonadotropin on TGFß
receptor mRNAs was also time dependent. The maximal effect of the
gonadotropin was observed after 6 and 4 h of treatment on TGFßRI
(Fig. 4B) and TGFßRII (Fig. 5B) mRNA levels, respectively.
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indicate that, as expected, hCG
treatment increased the levels of TGFßRI and -RII, but (pre)treatment
with aminogluthetimide plus hCG did not suppress hCG-stimulated
TGFßRI and RII expression. Together, these observations suggest that
the stimulatory effect of LH/hCG on TGFß receptors is probably not
mediated by steroid hormones, particularly by testosterone.
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Discussion |
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For both TGFßRI and TGFßRII, we detected one form of mRNA as well as one major band of protein using Northern and Western blotting analyses, respectively. In the cross-linking experiments, TGFß appears to bind not only to TGFßRI, TGFßRII, and betaglycan, but also to other (membrane) binding proteins. The nature of these other proteins is unknown. It will be of interest to identify these proteins as 1) they may modulate TGFß action at the Leydig cell level; and 2) they appear to be also regulated, as are the TGFß receptors, by LH/hCG (see below). Together, the presence of TGFß receptors identified through specific mRNAs and proteins detected with specific antibodies and through their binding ability (cross-linking experiments) indicates that these receptors are functional and are probably involved in the previously reported TGFß modulatory action on steroid hormone formation in Leydig cells (for reviews, see Refs. 2, 3). Furthermore, identification of the expression in Leydig cells of Smads known to be involved in TGFß action reinforces the functional aspect of TGFß receptors in the testicular cells.
One of the major observations made in the present paper is related to the positive regulatory action of LH/hCG on TGFß receptor expression in Leydig cells. Such an effect was more specifically observed on TGFßRI and TGFßRII. The hormone stimulated the expression of TGFßRI and -RII at both the mRNA and protein levels. Such a hormonal effect is probably mediated by the cAMP/protein kinase A pathway, as the LH/hCG action on TGFß receptors was mimicked by 8-bromo-cAMP (our unpublished data). That TGFßRI and -RII were positively regulated by LH/hCG at both the mRNA and protein levels would suggest that the hormone may act at a transcriptional level, although it remains to be determined whether LH/hCG increases TGFß receptor type I and II gene transcription and/or receptor mRNA stability. Finally, as Leydig cell stimulation with LH/hCG resulted in the production of steroid hormones, particularly testosterone, the possibility that the action of LH/hCG on the receptors is mediated by these hormones was examined. Such a possibility appears not to occur, as inhibition of the production of steroid hormone after aminogluthetimide treatment did not reduce the action of LH/hCG on TGFß receptors. By contrast, our data indicate that aminogluthetimide enhances the action of LH/hCG on TGFß receptors, evaluated through the cross-linking experiments. Based on such an observation, one may speculate that testosterone (or other Leydig cell steroid hormones) inhibits TGFß receptors. However, although a negative regulatory action of testosterone on TGFß receptors has been reported in rat extragonadal tissues such as the ventral prostate (33), such an effect of steroid hormone in testicular Leydig cells remains to be demonstrated. As testosterone (and other Leydig cell steroids) appears not to mediate LH/hCG action on TGFß receptors, the possibility exists that a mediating effect might be exerted by local signaling molecules, such as testicular growth factors and cytokines (2, 3).
In extragonadal tissues, TGFß receptors have been reported to be
modulated by different factors and/or in different physiological and
pathological conditions. For example, TGFß receptor expression has
been shown to depend on several cell parameters, such as energy
metabolism (34, 35), and the types of the extracellular matrix
components (36, 37). Also, TGFß receptors were decreased in
pathological situations, such as in tumor cells (38, 39, 40, 41, 42), and increased
in other pathologies, such as fibrosis (43) and Alzheimer’s disease
(44). Finally, hormones, specifically those acting though nuclear
receptors, may modulate TGFß receptor expression. Specifically,
vitamin D (45), estradiol administrated during fetal life (46),
testosterone (47), and finasteride (48) (an inhibitor of
the conversion of testosterone to 5-dihydrotestosterone) have been
shown to affect TGFß receptor expression. The present data are to our
knowledge the first demonstration that hormones such as gonadotropin
(acting via the cAMP/protein kinase A pathway) up-regulate TGFß
receptor expression in terms of both proteins and mRNAs in the male
gonad. In the female gonad, Roy and Kole (49, 50) reported, using
immunohistochemistry and immunoblotting approaches, that LH and FSH
enhance TGFßRII protein in hamster and human ovaries. In these
reports the TGFß receptors in the ovary were not studied at the mRNA
level. In addition, by contrast to our present findings, TGFßRI
protein was not affected by gonadotropins in the ovary.
Finally, in addition to the regulatory action of the gonadotropins on TGFß receptors, it will be interesting to determine in the future whether the gonadotropin may affect other components of the TGFß transducing system, such as Smads, which are present in Leydig cells (our present data).
Together, the numerous observations from different laboratories
showing that TGFß antagonizes LH/hCG steroidogenic action in Leydig
cells (for reviews, see Refs. 2, 3) coupled to our present findings
demonstrating that LH/hCG increases the expression of functional TGFß
receptors, and thus potentially TGFß action, support the concept of
the existence of a short loop between the hormone and the growth factor
at the Leydig cell level. It is tempting to speculate that LH/hCG may
use the TGFß system to end or reduce its own steroidogenic action or
that of other growth factors that enhance LH/hCG stimulated-steroid
hormone production in Leydig cells, such as epidermal growth
factor/TGF and insulin-like growth factor I (for reviews, see Refs.
2, 3). Indeed, in our laboratory, we have previously shown that
TGFß antagonizes the stimulatory action of epidermal growth
factor/TGF (51) and insulin-like growth factor I (52) on
LH/hCG-induced testosterone formation. Such interactions between the
growth factors and the gonadotropin may occur to modulate the
intratesticular levels of testosterone required for correct
spermatogenesis (1). However, besides its action on steroid hormone
formation, TGFß exhibits other activities, particularly on cell
proliferation, immune response, extracellular matrix component
formation, and cell death, and therefore one cannot exclude the
possibility that the gonadotropin, through the modulation of TGFß
receptors, may indirectly control such activities in the testis.
In summary, using porcine cultured Leydig cells as a model, we have characterized in these testicular cells the mRNAs and functional binding proteins for TGFß receptors and have shown that such receptors are under the control of the endocrine system through LH/hCG action.
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Acknowledgments |
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Received November 11, 1999.
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