Deregulation of anti-Mullerian hormoneBMP and transforming growth by lee92256

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									                                                                              Endocrine-Related Cancer (2008) 15 217–227



Deregulation of anti-Mullerian
hormone/BMP and transforming growth
factor-b pathways in Leydig cell lesions
developed in male heterozygous multiple
endocrine neoplasia type 1 mutant mice
Nader Hussein1,2, JieLi Lu1,2, Huguette Casse1,2, Sandra Fontaniere1,2,
                                                                  `
                   3
Anne-Marie Morera , Se ´ verine Mazaud Guittot 3, Alain Calender1,2,5,
Nathalie Di Clemente4 and Chang X Zhang1,2,5
1
          ´                                           ´
 Universite Claude Bernard Lyon, Lyon, France; Faculte de Me                  ´
                                                            ´decine, Universite Lyon 1, Lyon F-69003, France; Laboratoire
  ´ ´          ´
Genetique Moleculaire, Signalisation et Cancer, 8 Avenue Rockefeller, Lyon F-69373, France
2
 CNRS UMR5201, Lyon F69373, France
3
                        ˆ
 INSERM-INRA U418, Hopital Debrousse, Lyon F-69322, France
4
 INSERM, U782, Clamart F-92140, France; Univ Paris-Sud, UMR-S0782, Clamart F-92140, France
5
 Groupe d’Etude Recherche, CNRS No 2906, Lyon, France
                                                               ´ne         ´
(Correspondence should be addressed to C X Zhang, Laboratoire Ge ´tique Moleculaire, Signalisation et Cancer, CNRS,
                ´     ´                ´
UMR5201, Faculte de Medecine, Universite Claude Bernard Lyon 1, 8 Avenue Rockefeller, 69373 Lyon, France;
Email: zhang@sante.univ-lyon1.fr)
N Hussein and J Lu contributed equally to this work



Abstract
Multiple endocrine neoplasia type 1 (MEN1) results from the mutation of the predisposing gene,
MEN1. Heterozygous Men1 mutant mice previously generated by several laboratories, including
ours, mimic largely MEN1 pathology. Interestingly, our heterozygous Men1 mutant mice exhibit not
only the endocrine tumours commonly seen in MEN1 patients, but also Leydig cell tumours (LCT) with
high frequency, accompanied systematically by loss of the wild-type Men1 allele. As there exists a
similarity of tumour phenotype between these mice and those mutated for the components of anti-
Mullerian hormone (AMH)/bone morphogenic protein (BMP) pathway belonging to transforming
growth factor-b (TGF-b) family, we investigated the expression and the activity of this pathway, known
to have an important biological role in Leydig cells. Here, we report that the expression of AMH
receptor type 2 is reduced in Men1 LCTs. Both immunostaining and western blot analyses also
demonstrate a markedly decreased nuclear expression of Smad1, 3, 4 and 5 in the tumours. More
interestingly, we show that the reconstituted menin expression in Men1-deficient Leydig cells derived
from LCTs can significantly increase the transcriptional activity of a BMP pathway target promoter,
XVent2. Furthermore, we found that the expression of p18, p27 and cyclin dependant kinase 4 (Cdk4)
, targets of TGF-b pathways, is altered in the Leydig cell lesions. Our data provide the evidence of the
deregulation of AMH/BMP and TGF-b pathways in mouse Men1 LCTs, highlighting their involvement
in tumorigenesis of Leydig cells due to Men1 inactivation.
Endocrine-Related Cancer (2008) 15 217–227



Introduction                                                             of multiple endocrine tumours of the parathyroids,
                                                                         pancreas, anterior pituitary and adrenal cortex (MEN1,
The patients with multiple endocrine neoplasia type 1                    OMIM 131100). Other endocrine and non-endocrine
(MEN1), a hereditary syndrome transmitted with an                        tumours, such as forgut carcinoids, follicular thyroid
autosomal dominant trait, predispose to the occurrence                   tumours, angiofibroma, lipoma and smooth muscle
Endocrine-Related Cancer (2008) 15 217–227                                                                          DOI: 10.1677/ERC-06-0046
1351–0088/08/015–217 q 2008 Society for Endocrinology Printed in Great Britain         Online version via http://www.endocrinology-journals.org
N Hussein, J Lu et al.: AMH/TGF-b pathways in Leydig cell tumours

tumours, can also be associated with the disease            Sertoli cells, whereas the gene disruption of either Amh
(Thakker 1995). Germ line mutations in the MEN1             or Amhr2 in mouse resulted in Leydig cell hyperplasia,
gene have been detected in the majority of familial         indicating an essential effect of AMH pathway on the
MEN1 cases (Agarwal et al. 1997, Bassett et al. 1998,       control of Leydig cell proliferation. Moreover, the
Giraud et al. 1998), and somatic mutations have also        double inhibin-a/Amh-deficient mutant mice gave rise
been found in several types of sporadic endocrine           to the earlier development of granulosa/Sertoli cell
tumour, especially sporadic parathyroid adenomas            tumours and multifocal Leydig cell neoplasia (Matzuk
(Farnebo et al. 1998), gastrinomas and insulinomas          et al. 1995). Since previous works have demonstrated
(Zhuang et al. 1997). Both germ line and sporadic           the physical and functional interactions between menin
mutations show a typical loss of function profile,           protein, encoded by the MEN1 gene, and the effectors
with different types of mutation detected along             of TGF-b/BMP pathway, Smad1, 3 and 5, in various
the whole coding sequence (Schussheim et al. 2001,          cell types and tissues (Kaji et al. 2001, Sowa et al.
Wautot et al. 2002). However, these observations do not     2003), we decided to investigate whether there is a
establish any genotype–phenotype correlation. The loss      common molecular basis among LCTs developed in
of heterozygosity frequently observed in MEN1               heterozygous Men1 mice and those found in mice with
tumours supports the hypothesis that the MEN1 gene          deficiency in AMH pathway. Here, we report that the
acts as a tumour suppressor in affected cells (Larsson      deregulation of AMH/BMP and TGF-b pathways is
et al. 1988, Bystrom et al. 1990, Debelenko et al. 1997).   evidenced in the LCTs developed in heterozygous
   In order to study the mechanisms involved in             Men1 mice, accompanied by the altered expression of
multiple endocrinopathy related to MEN1 gene                its target genes. Our data thus provide insights into the
inactivation, several laboratories, including ours,         molecular basis of the physiopathological consequence
have generated Men1 mutant mice (Crabtree et al.            of Men1 inactivation in Leydig cells.
2001, Bertolino et al. 2003a,b, Biondi et al. 2004).
The heterozygous Men1 mutant mice develop, starting
at around 12 months of age, multiple endocrine              Materials and methods
tumours. To our surprise, in addition to the endocrine
tumours similar to those commonly described in              Men1 mutant mice and genotyping
MEN1 patients, we observed gonadal sex-cord stromal         Mice carrying an inactivated Men1 allele (Men1C/T)
tumours with high frequency in our heterozygous             were generated as described previously (Bertolino
Men1 mutant mice, including Leydig cell tumours             et al. 2003b). All animal experiments were conducted
(LCTs) in males and sex-cord stromal cell tumours of        in accordance with accepted standards of human
the ovary in females (Bertolino et al. 2003a).              animal care and were approved by the International
Interestingly, Loffler et al. (2007) have recently also      Agency for Research on Cancer’s Animal Care and
reported the occurrence of gonadal sex-cord stromal         Use Committee. PCR analyses were performed to
tumours in their heterozygous Men1 mutant mice. In          determine the presence of the wild-type and targeted
our hands, the loss of heterozygosity (LOH) were            Men1 alleles as described previously (Bertolino et al.
systematically detected in these tumours, suggesting a      2003b).
direct link between Men1 gene inactivation and the
development of these tumours in Men1C/T mice
                                                            Cell lines and transfection assays
(Bertolino et al. 2003a, Hussein et al. 2007). However,
although a case of LCT has been reported in a MEN1          MA10 (mouse Leydig cell line) cells were cultured in
patient (Ibarguren et al. 1992), the clinical significance   Waymouth’s MB 752/1 medium (Invitrogen Inc.)
of this observation in mouse Men1 model remains to be       supplemented with 20 mM HEPES, 15% (v/v) horse
elucidated.                                                 serum (Invitrogen) and 25 mg/ml gentamycin at 37 8C
   Interestingly, we have noticed that several mouse        with 5% CO2. Mouse Leydig tumour cell (MLTC) cells
models with deficiency of transforming growth                were cultured in RPMI 1640 medium (Sigma–Aldrich
factor-b (TGF-b) family components present similar          Corp.) supplemented with 10% fetal calf serum. Mouse
tumour phenotypes to the above-mentioned gonadal            embryonic fibroblasts were grown in DMEM
tumours, including knockout mice for inhibin-a, anti-       containing 25 mM glucose and supplemented with
Mullerian hormone (AMH), and its type 2 receptor,           10% (v/v) fetal calf serum, 100 units/ml penicillin,
namely AMHR2 (Matzuk et al. 1992, 1995, Behringer           100 mg/ml streptomycin, 2 mM L-glutamine and
et al. 1994, Mishina et al. 1996). The inhibin-a mutant     100 mM b-mercaptoethanol (Sigma), and LCT10 cells
mice developed gonadal tumours affecting principally        were grown in the same medium at 37 8C with 5%


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                                                             Endocrine-Related Cancer (2008) 15 217–227

CO2, except supplemented with 15% (v/v) horse             RT-PCR and northern blot analysis
serum, 2.5% (v/v) fetal bovine serum (Sigma) and          For the detection of Amhr2, total RNAs were extracted
without b-mercaptoethanol. Chinese hamster ovary          from cells or testis, with single-step RNA extraction
(CHO) cells were grown in standard DMEM. Cells            system (TRI-REAGENT, Sigma). cDNA was
were transfected using lipofectamine 2000 (Invitrogen)    synthesised with Superscript II reverse transcriptase
according to manufacturer’s instructions.                 (Invitrogen). The expression of Amhr2 and 3b-hydroxy
                                                          steroid dehydrogenase and actin transcripts was studied
Plasmids                                                  by RT-PCR using primers and conditions described
The expression vectors containing MEN1 cDNA               previously (Hussein et al. 2007). For northern blot
inserted in pCI-neo vector (Promega) either in sense,     analysis, the standard conditions (Racine et al. 1998)
referred to as pCI-M1S, or in opposite direction, as      were used to detect Amhr2 transcript with a probe
pCI-M1AS were used for menin reconstitution experi-       generated from the above-described RT-PCR. For the
ments (Wautot et al. 2000). Furthermore, two MEN1         detection of Smad transcripts, purified Leydig cells
mutations pCI-1384delAGG and pCI-Arg415ter were           were prepared and checked from Swiss male CD-1 mice
also generated (Hussein et al. 2007). XVent2-luciferase   at 8 weeks of age as previously described (Racine et al.
plasmid has been kindly provided by Dr K W Cho            1998), and total RNAs were extracted from purified
(University of California at Irvine, USA). The            Leydig cells and subjected to RT-PCR analysis using
construct containing Amhr2 cDNA inserted in               primers shown on Supplementary Table 1, which can be
pCDM8 vector (Gouedard et al. 2000) was used for          viewed online at http://erc.endocrinology-journals.org/
positive control of AMHR2 detection. The whole            supplemental/. Briefly, reverse transcription was per-
length MEN1 cDNA was cloned in pGex-4T1                   formed in a total of 20 ml with the First-Strand cDNA
(Amersham Biosciences) for glutathione-S-transferase      Synthesis Kit for RT-PCR (Roche Diagnostics) using
(GST) pulldown assay. The construct was verified by        1 mg RNA, AMV reverse transcriptase and random
DNA sequencing.                                           primers p(dN)6 as recommended by the manufacturer.
                                                          PCR was carried out in 25 ml PCR buffer containing
Protein extraction, immunoprecipitation, GST              3 mM MgCl2, with 2 ml appropriate cDNA, 400 nM
pulldown and immunoblotting analysis                      forward- and reverse-specific primers, 2 mM of each
                                                          dNTP and 0.5 U Taq polymerase. The PCR protocol
Enriched nuclear and cytoplasmic protein fractions        used an initial denaturation step at 95 8C for 3 min
from cells were prepared and analysed by western          followed by 30 cycles of denaturation at 95 8C for 45 s,
blotting as described previously (Wautot et al. 2000).    annealing at 60 8C for 45 s and elongation at 72 8C for
The following used primary antibodies were purchased      45 s. Amplified products were analysed by electro-
from Santa-Cruz Biotechnology (Santa Cruz, CA,            phoresis on a 10% polyacrylamide gel containing
USA): anti-menin (C19, 1:7500), anti-Smad1                tris-borate-EDTA (TBE) buffer and stained with
(1:1000), anti-Smad4 (1:1000) and anti-Smad5              ethidium bromide.
(1:750) antibodies. Anti-phospho-Smad1 polyclonal
antibody (1:1000) was purchased from Upstate
                                                          Histopathological and immunohistochemical
Biotechnology Inc. (Lake Placid, NY, USA), and
                                                          analyses
anti-actin monoclonal antibody from ICN (1:50 000,
Aurora, CA, USA), HRP-secondary antibodies from           Testes were collected from wild-type and heterozygous
Amersham. Anti-AMHRII antibody was used as                Men1 mutant mice and fixed in 4% buffered formalin
described previously (Gouedard et al. 2000).              for at least 24 h, followed by dehydration and paraffin
   For co-immunoprecipitation experiments, cells were     embedding. Histopathological analysis was carried out
treated 2 h with either AMH (71.4 nM), BMP2               on 3 mm sections stained with haematoxylin–eosin.
(10 nM), or not treated. Immunoprecipitation was          Immunohistochemical staining was performed as
carried out according to the protocol described           described previously (Bertolino et al. 2003a), using
previously (Gouedard et al. 2000). For GST pulldown       antibodies against menin (C19, 1:500), anti-p18
assay, purified GST-menin (GM) protein or GST              (1:3000), anti-p27 (1:1000), anti-pSmad1 (1:1000),
control protein (G) was incubated with cell extracts      anti-Smad1 (1:1000), anti-Smad3 (1:1000), anti-
prepared from MA10 cells treated 2 h with either AMH      Smad4 (1:1000), anti-Smad5 polyclonal (1:1000) and
(71.4 nM) or BMP2 (10 nM), or not treated, the            anti-Cdk4 (1:1000, Santa-Cruz Biotechnology)
co-sedimented proteins were revealed in western blot      antibodies. In all these analyses, the control without
using standard conditions.                                primary antibody was systematically included to rule


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N Hussein, J Lu et al.: AMH/TGF-b pathways in Leydig cell tumours

out the non-specific staining due to secondary
antibodies (data not shown).

Dual-luciferase reporter assay
For the reporter assay, 1 mg/well of XVent2 plasmid,
together with a co-reporter vector expressing Renilla
luciferase driving by a thymidine kinase promoter
(phRL-TK, Promega) at a ratio of 1:100 (10 ng) were
transfected in triplicate in LCT10 cells using six-well
plates by lipofectamine 2000. Menin’s effect on XVent2
reporter was assessed by the transfection of 1 mg/well of
the vectors expressing either wild-type or mutant menin.
Cells were incubated at 37 8C and harvested 48 h after
transfection. Cell lysates were assayed for luciferase
activity using the Dual-Luciferase Reporter Assay
System (Promega) in a luminometer tube (Dynatech
Laboratories Inc., Chantilly, VA, USA) according to
manufacturer’s instructions. Luciferase activity was
measured and normalised for transfection efficiency          Figure 1 Altered expression of the Amhr2 gene in mouse Men1
                                                            Leydig cell tumours. (A) RT-PCR analyses of testes from
with the activity of a co-transfected thymidine kinase      Men1C/T mutant mice at 20 months of age (TTU1–TTU5) and
promoter/Renilla luciferase reporter.                       from age-matched wild-type mice (T1WT–T2WT) simul-
                                                            taneously for both Amhr2 and 3b-Hsb transcript. MEF and
                                                            MA10 cells were used for negative and positive controls
                                                            respectively of the RT-PCR analysis. (B) Representative
Results                                                     northern blot analyses of testes from Men1C/T mutant mice at
                                                            20 months of age (TTU1–TTU2) and from age-matched wild-
Reduced AMHR2 expression in Men1 Leydig                     type mice (T1WT–T4WT) for Amhr2 transcript. RNA loading is
cell tumours                                                monitored with a probe for Gapdh transcript. (C) Detection of
                                                            AMHR2 by western blot analysis. Total protein extracts (20 mg)
To investigate whether there is a deregulation of AMH       from two testes isolated from wild-type Men1 mice at 20 months
pathway in Leydig cell lesions developed in male            of age (T1WT and T2WT) and four testes from Men1C/T mutant
                                                            mice at 20 months (TTU1 – TTU4) were used for western blot
heterozygous Men1 mutant mice, we have firstly               analysis using anti-AMHR2 antibody. CHO* (transfected with
analysed the expression of Amhr2 transcript using           Amhr2 expression vector) and MA10 cells were used as
both semi-quantitative RT-PCR (Fig. 1A) and northern        positive controls. The protein loading was monitored by an anti-
                                                            actin antibody.
blot (Fig. 1B) in the testes from male heterozygous
Men1 mutant mice at 20 months of age. Surprisingly,
we found that, although Amhr2 is known to express           namely Smad1 and 5, in Leydig cell lesions from
specifically in normal adult Leydig cells, its expression    Men1 mutant mice. To make sure that Smad genes are
was downregulated in the testes from all tested male        expressed in normal mouse Leydig cells as previously
heterozygous Men1 mutant mice, invaded by a massive         reported (Jiao et al. 2002, Hu et al. 2003), we have
amount of Leydig cells. To know whether the                 carried out the detection of their transcripts by RT-PCR
expression of the Amhr2 gene is reduced at the protein      analysis using purified Leydig cells. Our data showed
level, we have checked its expression by western blot       that all the tested Smad genes are expressed in the latter
analysis. The results demonstrated that AMHR2 was           (Supplementary Fig. 1, which can be viewed online at
clearly reduced in the testes from tested male              http://erc.endocrinology-journals.org/supplemental/),
heterozygous Men1 mutant mice at 20 months of age
                                                            as well as in two different mouse Leydig tumour cell
(Fig. 1C), compared with those from the age-matched
                                                            lines MA10 and MLTC. To detect eventual alteration
control mice, especially considering that the tumours
                                                            of Smad expression in Leydig cell lesions developed in
are mainly composed of Leydig cells.
                                                            Men1 mutant mice, we have used immunostaining
                                                            analysis as it allows comparing more specifically their
Decreased expression and activities of Smads in             expression in normal Leydig cells with that in Leydig
mouse Men1 Leydig cell lesions                              cells lesions. To this end, immunostainings using both
We have further analysed the expression of the              anti-Smad1 and anti-phosphorylated Smad1 antibodies
receptor-regulated Smads of AMH/BMP pathway,                were performed on the testes respectively from male


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                                                                        Endocrine-Related Cancer (2008) 15 217–227

heterozygous Men1 mutant mice at 12 months (nZ5)                    enriched fractions of protein extracts using anti-
and 20 months of age (nZ5), and the age-matched                     phosphorylated Smad1 antibody, as we found the
control mice (nZ5 for each age group). The results                  expression of phosphorylated Smad1 greatly reduced
showed that Smad1 protein could be readily detected                 in the testes from the mutant mice, compared with
by immunohistochemistry using anti-Smad1 antibody                   those from the control mice (Fig. 2J).
in the cytoplasm and nucleus of Leydig cells, and also                 Immunostaining of Smad5 demonstrated that, in
in the seminiferous epithelium from the control mice                contrast to what observed in the testes from Men1 wild-
(Fig. 2A), similar to what has previously been reported             type mice (nZ5, Fig. 2G), its expression was virtually
(Jiao et al. 2002, Hu et al. 2003). On the contrary, its            absent in both Leydig cell hyperplastic lesions and
expression was detectable only in the cytoplasm, but                tumours from Men1 mutant mice (nZ5 for each group,
not in the nucleus in Leydig cell hyperplastic lesions              Fig. 2H and I). The results were confirmed by western
(Fig. 2B). Smad1 expression was further reduced in the              blot analysis of nuclear-enriched fractions of protein
cytoplasm and undetectable in the nucleus in Leydig                 extracts using anti-Smad5 antibody. As illustrated in
cells tumours (Fig. 2C). The similar result has been                Fig. 2K, nuclear Smad5 could be detected in none of
obtained when using an antibody recognising phos-                   the five mice tested except one (TTU4) where a trace of
phorylated Smad1, the activated form of Smad1 with                  nuclear Smad5 expression was found.
mainly nuclear localisation (Fig. 2D–F). The results                   Smad3, a menin-interacting protein like Smad1 and
were confirmed by western blot analysis of nuclear-                  5, is the receptor-regulated Smad of TGF-b pathway.




Figure 2 Reduced Smad1 and 5 expression in mouse Men1 Leydig cell lesions. Immunohistochemical analysis with anti-Smad1
(A–C), anti-phosphorylated Smad1 (D–F) and anti-Smad5 (G–I) antibodies were performed on testes sections from wild-type mice at
20 months of age (A, D and G), from Men1C/T mice at 12 months of age (B, E and H) and from Men1C/T mice at 20 months of age
(C, F and I). Insets show an amplified view of testis sections. The scale bars are 50 mm. (J and K) Western blot analysis of phospho-
Smad1 and Smad5 expression. Nuclear protein extracts (20 mg) from two testes isolated from wild-type Men1 mice (T1WT and
T2WT) and testes from six Men1C/T mice at about 20 months of age (TTU1, TTU2, TTU3, TTU4, TTU5 and TTU6) were
immunoblotted, and revealed respectively with anti-phospho-Smad1 and anti-Smad5 antibodies. Nuclear extracts from MA10 cells
treated with AMH were used as positive control. Protein loading was monitored by an anti-actin antibody. This is representative of
three independent experiments.



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N Hussein, J Lu et al.: AMH/TGF-b pathways in Leydig cell tumours

Since this pathway is involved in the development of                fraction, suggesting the decreased total expression of
gonadal sex-cord stromal tumours in inhibin-a mutant                Smad4 in Leydig cell tumour.
mice, we investigated the expression and the activity of               Taken together, our data indicate a reduced
Smad3 in Leydig cell lesions in heterozygous Men1                   expression and activity of Smad1, 3, 4 and 5 in
mutant mice. Immunohistochemistry analysis showed                   mouse Men1 Leydig lesions. In particular, the reduced
that Smad3 expression was found in the cytoplasm and                expression of Smads was more evident in the nucleus
nucleus of normal seminiferous epithelia and Leydig                 of Leydig cell lesions, indicating the reduction of
cells (nZ3; Fig. 3A), whereas it was undetectable in                active form of Smads.
the nucleus in Leydig cell hyperplastic lesions (nZ6)
nor in Leydig cell tumours (nZ4) from Men1 mutant                   Menin interacts with Smad1 and regulates
mice (Fig. 3B and C).                                               AMH/BMP target promoter in Leydig cells
   Smad4 is needed for the activity of both BMP and
                                                                    The above-revealed close correlation between
TGF-b receptor-regulated Smads. In order to know
                                                                    abnormal expression and activities of the components
whether Men1 inactivation can affect Smad4, we have                 of AMH/BMP pathway and menin inactivation in
examined its expression in Men1 Leydig cell lesions.                Leydig cells lesions led us to further analyse the
Immunostaining of Smad4 showed that it is expressed                 physical interaction between menin and Smad1 in
in normal seminiferous epithelia and in Leydig cells                Leydig cells, reported previously in osteoblasts (Sowa
both in the cytoplasm and nucleus (nZ4; Fig. 3D). As                et al. 2003). Our analysis showed that the endogenous
shown respectively in Fig. 3E and F, this expression                menin was co-immunoprecipitated by an antibody
was markedly reduced, but not totally absent, in Leydig             against Smad1 with protein extracts prepared from
cell hyperplastic lesions (nZ5) and Leydig cell                     mouse Leydig cell line, MA10 cells treated by BMP2
tumours (nZ5). Western blot analysis revealed a                     stimulation. Consistent with the previously reported
detectable but much weaker expression of Smad4 in                   work, Smad4 was detected in the same immuno-
the nuclear fraction of the testes from Men1 mutant                 precipitated protein complex (Fig. 4A). We noticed
mice, compared with the control mice (Fig. 3G),                     that such an interaction has not been observed upon
whereas Smad4 was undetectable in the cytoplasmic                   AMH stimulation in our condition (Fig. 4A). We have




Figure 3 Decreased Smad3 and 4 expression in mouse Men1 Leydig cell lesions. Immunohistochemical analysis with anti-Smad3
antibody (A–C), and anti-Smad4 (D–F) antibodies. (A and D) Testes from Men1 wild-type mice at 20 months of age. (B and E) Testes
from Men1C/T mutant mice at 12 months of age. (C and F) Testes from Men1C/T mutant mice at 20 months of age. Insets show an
amplified view of testis sections. The scale bars are 50 mm. (G) Western blot analysis of nuclear and cytoplasmic fractions of testes
from wild-type and Men1C/T mice respectively with an anti-Smad4 antibody. MA10 extracts were used as positive control. Protein
loading was monitored by anti-actin antibody. This is representative of three independent experiments.



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                                                                      Endocrine-Related Cancer (2008) 15 217–227

                                                                   Indeed, our data demonstrated that the incubation of
                                                                   nuclear extract from both BMP2- and AMH-stimulated
                                                                   MA10 cells with GST fusion protein containing full-
                                                                   length menin led to binding of endogenous Smad1
                                                                   (Fig. 4B), although its presence is rather weak in upon
                                                                   AMH stimulation. To further study the functional
                                                                   interaction of menin and Smads, we have investigated
                                                                   the effect of menin on the transactivation of target genes
                                                                   by this pathway. To this end, we have carried reporter
                                                                   assay using a widely used AMH/BMP target promoter
                                                                   (Gouedard et al. 2000), XVent2, in a Men1-deficient
                                                                   Leydig cell line, LCT10, generated in our laboratory
                                                                   from a Leydig cell tumour developed in a male
                                                                   heterozygous Men1 mouse. LCT10 cells keep basic
                                                                   features of Leydig cells, but do not carry the wild-type
                                                                   allele of the Men1 gene (Hussein et al. 2007). Moreover,
                                                                   they have higher expression levels of AMH/BMP
                                                                   pathway components than those found in mouse Men1
                                                                   Leydig cell tumours (data not shown), except Smad5,
                                                                   most likely due to the selection during their establish-
                                                                   ment. We found that menin re-expression in LTC10
Figure 4 Menin interacts with Smad1 and regulates AMH/BMP          cells enhanced significantly the transactivation of
target promoter XVent2-luciferase in Leydig cells. (A) Co-         XVent2 promoter by more than onefold increase,
immunoprecipitation of endogenous menin and Smad1.
Immunoprecipitation of 1 mg protein extracts from MA10 cells
                                                                   whereas the expression of two mutants of menin, either
stimulated respectively by BMP2, AMH and not stimulated was        with a truncation, pM1-Arg415ter, or with an in-frame
performed using anti-Smad1 antibody. Western blot analysis of      deletion, pM1-1384delAGG, did not exert such an
resulting immunoprecipitated proteins was done using anti-
menin and anti-Smad4 antibodies. Input; 20 mg MA10 total
                                                                   effect (Fig. 4C).
protein extracts. Note that menin can be co-immunoprecipitated
with Smad1 upon BMP2 stimulation, whereas Smad4 is
co-immunoprecipitated upon AMH and BMP2 treatment. This is         Deregulation of AMH/BMP and TGF-b targets in
representative of several independent experiments. (B) GST         mouse Men1 Leydig cell lesions
pulldown assay was performed on MA10 cell lysates. GST (G)
or GST-menin (GM) proteins immobilised on Sepharose beads          It is known that CDK inhibitors p18 and p27 are both
were incubated with lysates of MA10 cells treated (C) or not
(K) with AMH or BMP2. Co-sedimented Smad1 protein was
                                                                   regulated by AMH/BMP pathway and menin protein
detected with anti-Smad1 antibody. Coomassie Blue staining         (Ha et al. 2000, Karnik et al. 2005, Milne et al. 2005).
demonstrated that the same amount of GST and GST menin             Moreover, the mice with p18 ablation developed
proteins was used. Input represents 5% of the total cellular
lysate. (C) Effect of menin re-expression on the activity of
                                                                   Leydig cell tumours, while the double p18/p27
XVent2 promoter. To measure the activity of the XVent2-            knockout mice accelerated the development of Leydig
luciferase reporter gene in LCT10 cells co-transfected with        cells tumours with a shorter incubation period and
XVent2 promoter and vectors expressing either menin
(pCI-M1), or menin mutants (pCI-1384delAGG and pCI-
                                                                   higher penetrance (Franklin et al. 2000). Furthermore,
Arg415ter), or an empty vector (pCI-neo) and the vector with       we have recently shown that menin re-expression in
inversed Men1 cDNA insert (pCI-M1AS), were analysed using          Men1-deficient LCT10 cells increased the expression
dual-luciferase reporter assay. All results are expressed as
meanGS.D. Cells were transfected with the indicated plasmids
                                                                   of both p18 and p27 (Hussein et al. 2007). To analyse
in triplicate of at least five independent experiments. *P!0.005.   their expression in mouse Men1 Leydig cell lesions, we
                                                                   performed immunohistochemical analysis using anti-
attempted to do the immunoprecipitation in a reverse               p18 and p27 antibodies. The result showed that nuclear
order, but since Smad1 has a molecular weight close to             staining of active forms of p18 and p27 was readily
50 kDa, similar to the heavy chain of the primary                  detectable in a substantial proportion of Leydig cells in
antibody, we failed to do this experiment even with                Men1 wild-type mice (Fig. 5A and D), whereas the
Exacta kit (Santa Cruz), likely due to the interference            same staining was greatly reduced in Leydig cell
of the primary antibody for WB detection and the                   hyperplastic lesions (nZ5; Fig. 5B and E) and totally
limited amount of interacting endogenous proteins. To              disappeared in all the tested Leydig cell tumours (nZ5;
confirm the physical interaction of menin and Smad1 in              Fig. 5C and F). Our data demonstrate that there is
MA10 cells, we have used GST pulldown assay.                       an inactivation of p18 and p27 in mouse Men1 Leydig


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N Hussein, J Lu et al.: AMH/TGF-b pathways in Leydig cell tumours




Figure 5 Altered expression of AMH/TGF-b target genes in mouse Men1 Leydig cell lesions. Immunohistochemical analysis with
respectively anti-p27 (A–C), anti-p18 (D–F) and anti-Cdk4 (G–I) antibodies. (A, D and G) Testes from Men1 wild-type mice at 20
months of age. (B, E and H) Testes from Men1C/T mutant mice at 12 months of age. (C, F and I) Testes from Men1C/T mutant mice at
20 months of age. Insets show an amplified view of testis sections. The scale bars are 50 mm. This is representative of three
independent experiments.


cell tumours where the Men1 gene is completely                    and several Smads, this finding revealed in the current
inactivated.                                                      study may represent the molecular basis implicated in the
   We have also checked the expression of Cdk4, a                 development of Leydig cell lesions in Men1 mutant mice.
known target gene of TGF-b pathway (Ewen et al. 1993,             Our results afford thus specific clues to the understanding
Serrano et al. 1993, Hirai et al. 1995). Immunohisto-             of the mechanisms of Leydig cell tumour development
chemical analysis demonstrated that Cdk4 was inten-               triggered by Men1 inactivation.
sively expressed in both Men1 Leydig cell hyperplastic               Indeed, the downregulation of Amhr2 in Leydig cell
lesions and tumours (Fig. 5H and I), compared with what           lesions is reminiscent of the molecular context existing
detected in the control mice (Fig. 5C), suggesting its            in Amh and Amhr2 mutant mice. Since menin is also
deregulation. In addition, we have analyzed the                   known to interact with the R-Smad proteins of TGF-b
expression of other factors involved in cell cycle                pathway and that Smad3 expression and its sub-cellular
progression, including cyclin A2, D2 and E. However,              location were altered in mouse Men1 Leydig cell
we did not find any obvious expression of these genes in           lesions, the more severe tumour phenotype observed in
Leydig cell lesions (data not shown).                             Men1 mutant mice, compared with that found in Amh
                                                                  or Amhr2 mutant mice, could result from the
                                                                  synergistic effects of the deregulated both AMH/BMP
                                                                  and TGF-b pathways due to menin inactivation. In fact,
Discussion                                                        Matzuk et al. (1995) have shown that the double Amh
The current study demonstrate the deregulation of                 and inhibin-a knockout mice did develop Leydig cell
AMH/BMP and TGF-b pathways, from the decreased                    tumours, whereas the latter is only a rare event in Amh
presence of its type 2 receptor AMHR2 to the reduced              single mutant mice. Our data confirmed thus their
nuclear expression of the terminal effecter Smad4, as             results and highlighted further that the components of
well as the altered expression of several target genes in         both AMH/BMP and TGF-b pathways are vital in the
mouse Men1 Leydig cell lesions. Considering the very              control of Leydig cell proliferation. On the contrary,
similar tumour phenotype observed in Amh and Amhr2                the lack of Sertoli cell lesions in Men1 mutant mice
knockout mice and the known interaction between menin             may indicate that the inhibin-a pathway is not


224                                                                                                 www.endocrinology-journals.org
                                                                  Endocrine-Related Cancer (2008) 15 217–227

completely affected in Men1 inactivation, albeit the           one of the key factors that control the transition of cell
reduced Smad3 expression, revealing a cell type-               cycle from G1- to S-phase, and has previously been
specific role of menin in TGF-b pathway in testis               shown to be a target of TGF-b pathway in Leydig cells
interstitial tissue.                                           (Cipriano et al. 2001). Furthermore, Rane et al. (1999)
   The downregulation of Amhr2 observed in mouse               have reported that mice deficient for Cdk4 manifested a
Men1 Leydig cell tumours is rather surprising, as menin        reduced Leydig cell population, while mice expressing
is so far only known to interact with the down-stream          activated Cdk4 developed Leydig cell hyperplasia.
R-regulated Smads. We have failed to detect the direct         Recently, Ratineau et al. (2004) have also demon-
regulation of menin on a 350 bp proximal Amhr2                 strated that knockdown of menin expression resulted in
promoter by reporter assay (data not shown). Conse-            a significant increase of Cdk4 expression in IEC-17
quently, the mechanisms leading to the downregulation          cells. The current study suggests that the intensive
of Amhr2 remain elusive. However, it is known that the         expression of Cdk4 in mouse Men1 Leydig cell lesions,
downregulation of TGF-bR2 is widely observed in                together with the downregulation of p18 and p27 is a
human tumours, considered as a part of multi-step              part of molecular consequences of Men1 inactivation.
mechanisms of tumorigenesis allowing tumour                    It would be important to clarify whether and how its
progression. Interestingly, Ratineau et al. (2004) have        altered expression is caused by menin inactivation.
recently shown that knockdown of menin expression in a            AMH/BMP and TGF-b pathways are well known for
rat embryonic duodenal cell line, intestinal epithelial cell   their important biological role in Leydig cells. The
(IEC-17), led to the downregulated expression of TGF-          deregulation of these pathways due to Men1 inacti-
bR2. The reduced expression of Amhr2 observed in this          vation revealed by the current study should thus also
tumour model could result from the similar mechanism,          shed light onto the probable biological and endocrine
and thus could be an important molecular event in Leydig       functions of menin in Leydig cells. Furthermore,
cell lesions due to Men1 inactivation. The further work is     considering that menin may act, as recently reported
needed to elucidate the occurrence of Amhr2 down-              (Dreijerink et al. 2006), as a co-activator of nuclear
regulation in mouse Men1 Leydig cell lesions.                  receptor-mediated transcription and that Leydig cells
   Several studies using cellular models have pre-             are among androgen target cells, the study of menin’s
viously shown the physical and functional interaction          role in Leydig cells could be of importance for
between menin and Smad proteins, including Smad1, 3            understanding how the Men1 gene functions in cells
and 5, playing an important role in the control of cell        where nuclear receptor-mediated transcription plays a
proliferation and hormone production in pituitary and          crucial role in cell proliferation control.
parathyroid cells (Kaji et al. 2001, Lacerte et al. 2004,
Sowa et al. 2004b), and in osteoblasts (Sowa et al.
2003, 2004a). Importantly, our data demonstrated that          Acknowledgements
these functional and physical interactions also exist in       We thank Marie-Pierre Cros and Dominique Galendo
Leydig cells, indicating that menin’s biological and           for the maintenance of the mouse colonies, Christine
onco-suppressive role in Leydig cells may imply a              Carreira for technical assistance, Dr Zhao-Qi Wang
similar molecular mechanism than that observed in the          (International Agency for Research on Cancer, Lyon,
above-mentioned endocrine cells. Interestingly, our            France) for scientific discussion, and Dr KW Cho
data also revealed that the expression of target genes of      (University of California at Irvine, USA) for providing
AMH/BMP and TGF-b pathways, including that of                  XVent2-luciferase plasmid. This study was supported
p18, p27 and Cdk4, were deregulated, suggesting that           by the Association pour la Recherche contre le Cancer,
the alteration of the components of these two pathways                                                    ˆ
                                                               France, the Ligue contre le Cancer du Rhone and de la
in Leydig cells may resulted in the dysfunction of their       Loire. During this study, N Hussein was the recipient
regulation on proteins involved in cell cycle control.         of fellowships from the Association pour la Recherche
We have recently showed that menin re-expression in            contre le Cancer, France, and the Lebanese University.
Men1-deficient Leydig cells resulted in a cell cycle            There is no conflict of interest that would prejudice the
blockade from G1- to S-phase transition, and                   impartiality of this scientific work.
augmented the expression of p18 and p27 (Hussein
et al. 2007). The downregulation of p18 and p27 in
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