TEX11 Modulates Germ Cell Proliferation by Competing with Estrogen Receptor β for the Binding to HPIP

Abstract

Klinefelter syndrome (KS), characterized by the presence of more than one X-chromosome in men, is a major genetic cause of male infertility. Germ cell degeneration in KS patients is thought to be the consequences of overexpression of some genes on the X-chromosome. However, the identity of these genes and the underlying mechanisms remain unclear. Testis-expressed 11 (TEX11) is an X-chromosome-encoded germ-cell-specific protein that is expressed most abundantly in spermatogonia and early spermatocytes in the testes. In our search for TEX11-interacting partners using the yeast two-hybrid system, we identified hematopoietic pre-B cell leukemia transcription factor-interacting protein (HPIP), which anchors estrogen receptors (ER) to the cytoskeleton and modulates their functions. We found that mouse spermatogonial stem cells expressed Tex11, Hpip, and Esr2 but not Esr1. In cultured cells, TEX11 competed with ERβ for binding to HPIP. Upon treatment with 17β-estradiol or an ERβ agonist diarylpropionitrile, TEX11 promoted the nuclear translocation of ERβ and enhanced its transcriptional activities. On the other hand, TEX11 suppressed the nongenomic activities of ERβ in the cytoplasm, as indicated by reduced phosphorylation of AKT and ERK signaling molecules. Overexpression of TEX11 in mouse germ-cell-derived GC-1 and GC-2 cells suppressed the cell proliferation and the expression of cFos, Ccnd1, and Ccnb1 that were stimulated by 17β-estradiol or diarylpropionitrile and elevated the expression level of the proapoptotic Bax gene. The negative effect of TEX11 on cell proliferation suggests that increased expression of TEX11 in the germ cells may partially contribute to the spermatogenic defect observed in KS patients.

The mammalian sex chromosomes play important roles in the development and maintenance of the male reproductive system. In addition to the SRY gene on the Y-chromosome that dictates testis formation (1), the sex chromosomes are enriched for genes that are expressed exclusively or predominantly in the testis (2–5). As a consequence, sex chromosome abnormalities, such as Y-chromosome microdeletion and Klinefelter syndrome (KS), are major genetic causes of male infertility (6, 7). KS is characterized by the presence of more than one X-chromosome in a man, who typically has a 47,XXY karyotype (8). KS patients contain a normal number of germ cells in their gonads at birth, but the germ cells degenerate soon afterward, resulting in Sertoli-cell-only syndrome in most adult patients (9). The demise of germ cells in KS patients is thought to be caused by the increased expression of some genes from the extra X-chromosome that is reactivated during early germ cell development (10). However, the identity of these culprit genes and the underlying mechanisms remain unknown.

Tex11 is one of the X-linked spermatogenesis genes isolated from a mouse testis subtraction library enriched for germ-cell-specific genes (5). It was also picked up in a recent search for interacting proteins of Nijmegen breakage syndrome 1, a component of the meiotic recombination 11 complex that is involved in the double strand break during meiosis (11). Tex11 encodes a 104-kDa protein with no recognizable functional domain except a tetratricopeptide repeat motif that mediates protein-protein interaction (12). Northern blot analyses showed Tex11 signals in the testis only, whereas RT-PCR detected additional expression of Tex11 in embryonic ovaries (5, 11). In adult mouse testes, the testis-expressed 11 (TEX11) protein first appears in both the cytoplasm and the nuclei of type B spermatogonia, reaches the highest expression level in zygotene spermatocytes, and diminishes to background levels in late pachytene spermatocytes (11). In zygotene and early pachytene spermatocytes, distinct TEX11 foci appear along synaptonemal complexes, implicating TEX11 in homologous recombination (13). Such a role was supported by the phenotype of Tex11-null mice, which exhibited spermatogenic arrest at late meiosis, accompanied by increased synaptic failure, reduced crossover, and aberrant chromosome segregation (13). Another Tex11 mutant strain with a hypomorphic allele also exhibited delayed repair of double strand breaks and decreased crossover formation, yet the fertility of the male mice did not seem to be affected (11). The molecular mechanism underlying TEX11's function in meiosis remains to be elucidated (13). The abundant presence of TEX11 in type B spermatogonia and early spermatocytes suggests a different role of TEX11 in earlier stages of germ cell development.

Estrogens are important hormones for the growth and development of many tissues, including those in the male reproductive system (14, 15). They mediate biological effects through two intracellular estrogen receptors (ER), ERα and ERβ, encoded by the ESR1 and ESR2 genes, respectively. Whether the G protein-coupled receptor (GPR)30 represents a third ER remains debatable (16, 17). ERα and ERβ have similar overall structures and share significant sequence homology, especially in the DNA-binding domain (∼97%) and the ligand-binding domain (∼59%) (18). They are expressed in many tissues and exhibit distinct yet overlapping expression patterns (19). The ER exert their effects through two different mechanisms (reviewed in Ref. 14). Upon binding to estrogens, an ER may function as a transcription factor to regulate the expression of its target genes (the genomic effects), either through direct binding to estrogen response elements in their promoters, or through protein-protein interactions to some transcription factor complexes. Alternatively, the ligand-receptor complex may act as a signaling protein to rapidly activate kinase-dependent signaling pathways in the cytoplasm (the rapid nongenomic effects), which may indirectly influence gene expression. Recently, the ER were found to be anchored to the cytoskeleton through their association with hematopoietic pre-B cell leukemia transcription factor (PBX)-interacting protein (HPIP) (20, 21). HPIP is a microtubule-associated protein that shuttles between the cytoplasm and the nucleus (22). It binds to all members of the PBX family of transcription factors and has been shown to inhibit the transcriptional activity of the E-protein 2A-PBX fusion protein produced from the t(1:19) translocation found in 25% of pediatric acute lymphoblastic leukemia (23). HPIP also modulates rapid nongenomic estrogen signaling by recruiting the Src kinase and the p85 subunit of phosphatidylinositol 3-kinase (PI3K) to form a complex with ERα that activates the AKT and MAPK signaling pathways, leading to enhanced cell growth, motility, and tumorigenesis (20, 21). The effect of HPIP on the transcriptional activity of ERα remains controversial. Manavathi et al. (20) observed down-regulation of an estrogen-responsive reporter gene in MCF7 breast carcinoma cells overexpressing HPIP, whereas Wang et al. (21) found enhanced expression of ERα targeted genes in similarly transfected MCF7 cells.

Here we report our study on the interplay between TEX11, HPIP, and ERβ. We found that mouse spermatogonial stem cells expressed Tex11, Hpip, and Esr2 but not Esr1. TEX11 modulated both the genomic and nongenomic effects of ERβ through its competition with ERβ for binding to HPIP. Overexpression of TEX11 in mouse type B spermatogonia-derived GC-1 cells as well as MCF7 cells enhanced the transcription of an estrogen-responsive reporter gene, but suppressed the AKT and MAPK signaling pathways, leading to decreased cell proliferation. Our results suggest that increased expression of TEX11 in the germ cells of KS patients may partially contribute to the germ cell death.

Results

HPIP is a TEX11-interacting protein

We used two baits containing the N-terminal (amino acids 1–393) and the C-terminal (amino acids 298–947) portion of TEX11, respectively, in a yeast two-hybrid system to isolate TEX11-interacting proteins from a human testis cDNA library (Fig. 1A). The N-terminal bait failed to yield any positive clones, whereas the C-terminal bait identified six positives, representing four independent clones, containing the HPIP gene sequence (GenBank accession no. NM_020524) (Fig. 1B). We next generated a series of deletions in the TEX11 C-terminal bait (Fig. 1A) as well as one smaller HPIP clone (Fig. 1B) and used the yeast mating assay to narrow down the interacting region in each protein (Fig. 1A and Supplemental Fig. 1, published on The Endocrine Society's Journals Online web site at http://mend.endojournals.org). For HPIP, a segment containing amino acids 509–631 retained the ability to interact with TEX11 (Fig. 1B). For TEX11, more than one third of the C-terminal region (amino acids 578–947) was required for the interaction (Fig. 1A). Further cutting down the segment into smaller fragments inevitably abolished its ability to interact with HPIP, suggesting that such interaction involves a specific tertiary structure of TEX11.

Fig. 1.

HPIP interacts with TEX11. A, Regions of TEX11 (indicated in amino acid residues) encoded by the original baits and subsequent deletion clones. The abilities of the regions to interact with HPIP, as determined by the yeast mating assay, are shown at right. B, Portions of HPIP encoded by the yeast two-hybrid clones and a deletion clone. In both A and B, the striped bars show the minimal regions required for interaction. C, Regions encoded by the various TEX11 expression vectors. D, Coimmunoprecipitation of HPIP and TEX11. COS7 cells were transfected with expression vectors for HA-tagged TEX11 or its derivatives and HPIP-Flag. The cell lysates were mixed with an anti-HA antibody (HA) or mouse IgG, and the immunoprecipitates were Western blotted with the antibodies against the epitopes indicated at right. The input lane contains 1/10 of the lysate used in the immunoprecipitation. E, Similar to D except that an anti-Flag antibody (Flag) was used in the immunoprecipitation.

We next confirmed the interaction between TEX11 and HPIP by expressing hemagglutinin (HA)-tagged TEX11 and Flag-tagged HPIP simultaneously in COS7 cells and testing the ability of the antibody against either epitope to bring down both proteins. In addition to the full-length TEX11, we generated three derivatives, D1, D2, and D3, which had deleted different regions of the C-terminal portion and were not expected to bind HPIP (Fig. 1C). Our results showed that the anti-HA antibody was able to bring down HPIP-Flag only when the full-length TEX11, but not the derivatives, was expressed in the same cells (Fig. 1D). In the reciprocal experiments, the anti-Flag antibody was able to bring down HPIP-Flag together with the full-length TEX11, but not its derivatives (Fig. 1E). Similar results were obtained when MCF7 cells were used (data not shown). Thus, HPIP and TEX11 were able to bind each other in cultured cells. Additional confocal images of MCF7 cells expressing both HA-TEX11 and HPIP-Flag also showed partial overlap of the signals, lending additional support for the interaction between the two proteins (Supplemental Fig. 2).

Mouse spermatogonial stem cells express Hpip, Tex11, and Esr2

HPIP has been previously shown to modulate the activities of the ER (20, 21). To investigate the possibility that TEX11 mediates germ cell growth through HPIP and ER, we determined the expression of these three entities in male germ cells. Previous studies have showed that spermatogonia in most mammals, including mice and humans, express ERβ but not ERα (15). We used fluorescence-activated cell sorting (FACS) to isolate spermatogonial stem cells (SSC), which express the surface marker Thy1 (24), from the testes of 7- to 8-d postpartum (dpp) mice (Fig. 2A). Of the cells in the Thy1⁺ fraction, approximately 89–92 and 80–85% stained positive for two additional SSC markers, epithelial cell adhesion molecule (EpCAM) (25) and GPR125 (26), respectively (Supplemental Fig. 3). RT-PCR results showed that the Thy1⁺ cells expressed Esr2, Gpr30, Tex11, and Hpip, but little Esr1 (Fig. 2B). The weak signal of Esr1 apparently came from somatic cell contamination in the Thy1⁺ fraction, as indicated by the weak signal of Cst12, which encodes the Sertoli cell marker cystatin 12 (27). On the other hand, the Thy1⁻ fraction was free of SSC as shown by the absence of another SSC marker, Plzf (28). It would be very difficult to obtain sufficient SSC to study in detail the interplay between TEX11, HPIP, and ERβ. We therefore performed subsequent experiments in established cell lines, including mouse type B spermatogonia-derived GC-1 and spermatocyte-derived GC-2 cells (29) as well as MCF7 breast carcinoma cells, which were used previously to study the association between ER and HPIP (20, 21). Our RT-PCR results indicated that GC-1 and GC-2 cells expressed Esr1, Esr2, Gpr30, Tex11, and Hpip, whereas MCF7 cells expressed ESR1, ESR2, GPR30, and HPIP, but not TEX11 (Supplemental Fig. 4A). Additional Western blot showed that these three cell lines expressed comparable levels of ERα and ERβ, whereas GC-1 and GC-2 expressed similar levels of TEX11 (Supplemental Fig. 4B).

Fig. 2.

Mouse spermatogonial stem cells express Tex11, Hpip, and Esr2 but not Esr1. A. FACS profiles of single-cell suspensions of 7- to 8-dpp mouse testes without (left) and with (right) the addition of the anti-Thy1 antibody. The left panel was used to determine the baseline for the Thy1⁻ cells. In the right panel, regions collected for the Thy1⁻ (A) and Thy1⁺ (B) fractions are indicated. B, RT-PCR analyses of gene expression in the Thy1⁻ and Thy1⁺ cells. Testis RNA from a 17-dpp mouse was used as a positive control. Two independent experiments were performed for the results shown.

TEX11 and ERβ compete for binding to HPIP

HPIP has been shown to interact with the ER through a nuclear receptor-interacting motif, LASLL, located at amino acids 615–619 (20). Because this motif resides within the region of HPIP that was found to be required for its interaction with TEX11, we investigated whether TEX11would compete with ERβ for binding to HPIP. We transfected constant amounts of pHPIP-Flag and pERβ-Myc, and increasing amounts of pHA-TEX11 into GC-1 cells, and monitored the levels of ERβ and TEX11 immunoprecipitated together with HPIP with the anti-Flag antibody. Transfection with increasing amount of pHA-TEX11 resulted in a dosage-dependent increase in the expression of HA-TEX11and the amount of HPIP-bound TEX11 (Fig. 3A and Supplemental Fig. 5). On the other hand, the HPIP-bound ERβ showed a dosage-dependent decrease, even though the total levels of ERβ in the cells remained the same. When the full-length TEX11 was replaced with the D2 derivative that could not bind HPIP, the amount of HPIP-bound ERβ remained unchanged (Fig. 3B). Our results thus indicate that TEX11 and ERβ compete for the binding to HPIP.

Fig. 3.

TEX11 and ERβ compete for HPIP binding. A, GC-1 cells were transfected simultaneously with constant amounts of pHPIP-Flag and pERβ-Myc and various amounts of pHA-TEX11. The cell lysates were treated with the anti-Flag antibody, and the immunoprecipitates were Western blotted with the antibodies against the epitopes indicated at right. Western blots of total inputs before immunoprecipitation are shown at the bottom. Three independent experiments were performed. The relative signal intensities of total TEX11 and coimmunoprecipitated TEX11 and ERβ are presented in Supplemental Fig. 5. B, Similar to A except that pHA-TEX11 was replaced with pHA-D2. Three independent experiments were performed.

TEX11 enhances the transcriptional activities of ERβ

Because HPIP tethers ER to the cytoskeleton and modulates their transcriptional activities (20, 21), we investigated whether the competitive binding of TEX11 to HPIP affected the genomic effects of ERβ. We first transfected GC-1 cells with pERβ-Myc together with either pHA-TEX11, pHA-D2, or the empty vector pXJ-HA. Western blot analyses on total cell lysates showed that treatment with 17β-estradiol (E2) had little effect on the overall expression levels of ERβ-Myc in the transfected cells (Fig. 4A). We then fractionated the cells and monitored the nuclear-cytoplasmic distribution of ERβ-Myc. Expression of TEX11 or D2 alone did not significantly change the nuclear-cytoplasmic distribution of ERβ (Fig. 4, B and C). E2 treatment caused an influx of ERβ into the nucleus. In cells overexpressing TEX11, the increase in the level of nuclear ERβ was much higher than that in cells transfected with either the empty vector or pHA-D2. In a separate experiment, we treated transfected GC-1 cells with the ERβ-specific agonist diarylpropionitrile (DPN) and followed the subcellular distribution of ERβ-Myc by immunofluorescence staining (Supplemental Fig. 6A). Quantification of the fluorescence signals showed again higher levels of nuclear ERβ in cells overexpressing TEX11 and treated with DPN (Supplemental Fig. 6B). Taken together, our results indicate that TEX11 promotes the nuclear translocation of ERβ upon estrogen treatment.

Fig. 4.

TEX11 stimulates the transcriptional activity of ERβ. A, E2 treatment has no effect on the overall expression of ERβ. GC-1 cells transfected with pERβ-Myc together with the empty vector pXJ-HA, pHA-TEX11, or pHA-D2 were treated with or without 10 nm E2 for 5 min before being harvested and subjected to Western analysis. TEX11 and ERβ were detected by antibodies against the HA and Myc epitope, respectively. B, TEX11 promotes nuclear localization of ERβ. Nuclear and cytoplasmic fractions of GC-1 cells, transfected and treated with E2 in the same manner as those shown in A, were Western blotted. Lamin A and α-tubulin serve as markers for the nucleus and the cytoplasm, respectively. C, Signal intensities of nuclear and cytoplasmic ERβ, shown in B, were determined and the ratios of nuclear and cytoplasmic ERβ are shown. D, TEX11 enhances the expression of an estrogen-responsive luciferase reporter gene. MCF7 or GC-1 cells transfected with pGL3-C3, pRL-TK, and 0.5 μg of expression vectors for the various TEX11 proteins were treated with 10 nm E2 (MCF7, black; GC1, gray) or 1 μm DPN (GC1, white) for 16 h before being harvested and assayed for luciferase activities. Relative activities of firefly luciferase and Renilla luciferase are shown. E, Estrogen antagonist ICI 182,780 counters the stimulating effect of E2 and DPN. Cells were cultured in the presence or absence of ICI 182,780 and analyzed similar to D. F, ERβ knockdown by shRNA. GC-1 cells were transfected with shRNA expression vectors targeting luciferase (shLuc) or ERβ (shEsr2) and Western blotted using antibodies against the various proteins. Untransfected cells were used as the control. G, ERβ knockdown reduces the expression of the luciferase reporter gene. GC-1 cells were transfected similar to those in D except that some were also transfected simultaneously with shEsr2. Three independent experiments were performed for all results shown. In C–E and G, the results shown represent the averages of three independent experiments. Statistical significance of the differences was determined using the paired t test and is indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

We next studied the effect of TEX11 on the transcriptional activity of ERβ using a firefly luciferase reporter gene driven by the estrogen-responsive promoter of the complement component 3 (C3) gene (30). We transfected MCF7 and GC-1 cells with the luciferase reporter vector pGL3-C3, the internal control pRL-TK plasmid, and the expression vector for the full-length or truncated TEX11 and measured the reporter activities in the transfected cells. E2 treatment for 16 h greatly enhanced the reporter activities in MCF7 cells as previously reported (20, 21), and such enhancement was readily blocked by the simultaneous presence of the estrogen antagonist ICI 182,780 in the media (Fig. 4, D and E; black bars). GC-1 cells exhibited similar responses to E2 and ICI 182,780 (Fig. 4, D and E; gray bars). However, in both GC-1 and MCF7 cells, overexpression of TEX11 caused further increases in the reporter activities that were not observed in cells expressing the truncated D2. Because E2 acts on both ERα and ERβ, we repeated the experiments in GC-1 cells using the ERβ-specific agonist DPN and observed similar transcriptional stimulation by TEX11 (Fig. 4, D and E; white bars).

To confirm that the transcriptional stimulation by TEX11 was mediated through ERβ, we knocked down the endogenous ERβ in GC-1 cells with an Esr2 short-hairpin RNA (shRNA) (shEsr2), which caused approximately 70% reduction in the level of ERβ, but not ERα or TEX11 (Fig. 4F). The ERβ knockdown was accompanied by a similar reduction in the DPN-stimulated transcription in cells transfected with the empty vector (Fig. 4G). Overexpression of TEX11 in the knocked-down cells failed to enhance the reporter activity as seen before, indicating that ERβ is involved in the transcriptional stimulation of TEX11.

Taken together, our results showed that overexpression of TEX11 promoted the translocation of cytoplasmic ERβ to the nucleus and enhanced its transcriptional activity.

TEX11 inhibits the nongenomic activities of ERβ

We also monitored the phosphorylation of AKT and ERK signaling molecules after short estrogen treatment to investigate whether TEX11 affects rapid nongenomic activities of ERβ. As expected, subjecting GC-1 cells to DPN for 10 min resulted in significant increases in the levels of both phosphorylated AKT and phosphorylated ERK (pERK) (Fig. 5A). The increases were attenuated by the expression of exogenous TEX11 in a dosage-dependent manner, whereas no attenuation in phosphorylation was observed in cells expressing a similar level of D2 (Fig. 5, A and B), indicating that TEX11 inhibits the nongenomic activities induced by DPN. To further show that the inhibitory effect of TEX11 was mediated through ERβ, we knocked down the endogenous ERβ in GC-1 cells and observed a significant reduction in the DPN-induced ERK phosphorylation (Fig. 5, C and D). Overexpression of TEX11 in the knocked-down cells did not cause a further decrease in the pERK level, indicating that TEX11 and ERβ share the same signaling pathway.

Fig. 5.

TEX11 suppresses AKT and ERK phosphorylation induced DPN. A, GC-1 cells transfected with increasing amounts (as indicated) of pHA-TEX11 or 1.0 μg pHA-D2 were treated with DPN for 10 min before being harvested and Western blotted with antibodies specific for the phosphorylated and unphosphorylated AKT and ERK. B, Relative signal intensities of the phosphorylated proteins as shown in A. C, GC-1 cells were transfected with pHA-TEX11 (or the empty vector) and shEsr2 and treated with or without DPN for 10 min before being harvested and Western blotted. D, Relative intensities of pERK signals as shown in C. The results represent the averages of three independent experiments, and statistical significance of the differences was determined using the paired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

TEX11 suppresses E2/DPN-stimulated cell proliferation

Because ERβ also modulates cell growth, we next studied the effect of TEX11 on the proliferation of GC-1 and GC-2 cells. We transfected the cells with the empty vector, pHA-TEX11, or pHA-D2, and cultured them in media containing bromodeoxyuridine (BrdU) with or without E2 or DPN for 10 h before harvesting them and counting the total cell numbers (Fig. 6A). All cells cultured in the presence of E2 or DPN had higher cell counts compared with those grown in its absence, indicating that estrogens stimulated cell growth. Simultaneous treatment with a MAPK/ERK kinase inhibitor PD98059 (PD) abolished the growth stimulation, implying the involvement of the ERK signaling pathway. In cells expressing TEX11, but not those expressing D2, the growth-stimulating effect of E2/DPN was significantly suppressed. Additional FACS analysis showed that the fractions of cells positive for both BrdU and HA were comparable for cells transfected with pHA-TEX11 or pHA-D2 and grown in the absence of estrogen as well as cells transfected with pHA-D2 and treated with estrogen (Fig. 6B). However, in cells transfected with pHA-TEX11 and treated with estrogen, the fraction of cells positive for both BrdU and HA was significantly reduced (Fig. 6, B and C), indicating slower growth of cells overexpressing TEX11. This was not due to differences in the transfection efficiency because the same batch of transfected cells was used for the various treatments (see Materials and Methods). Our results thus indicate that TEX11 suppresses estrogen-stimulated cell proliferation, and the suppression is mediated through its binding to HPIP because D2 lacks such an effect.

Fig. 6.

TEX11 Inhibits E2- or DPN-stimulated cell proliferation. A, GC-1 or GC-2 cells were transfected with the TEX11 or D2 expression vector and cultured in the presence of 10 μm BrdU with or without 10 nm E2, 1 μm DPN, or 10 μm PD for 10 h before being harvested and counted. B, The cells in A were subjected to FACS analysis to determine the cell fractions that were positive for both BrdU and HA. Two representative FACS distribution profiles are shown in C. In A and B, the results shown represent the averages of three independent experiments. Statistical significance of the differences was determined using the paired t test and is indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

TEX11 affects the expression of estrogen target genes

We further studied the effects of TEX11 on the expression of several downstream targets of estrogens (Fig. 7). c-Fos and Hras1 are protooncogenes that regulate cell growth and cellular transformation (31–33), Ccnd1 and Ccnb1 control cell cycle progression (32, 34, 35), and Bax and Bcl2 are proapoptotic and antiapoptotic genes, respectively (32, 35, 36). We found that DPN treatment of GC-1 cells for 6 h elevated the RNA levels of c-Fos, Ccnd1, and Ccnb1 over 4-fold, and overexpression of TEX11 in the cells significantly suppressed the increases (Fig. 7). Simultaneous PD treatment abolished the DPN-stimulated expression, indicating that the enhanced gene expression was mediated through nongenomic effects of the estrogen. On the other hand, DPN treatment suppressed the expression of Bax, Bcl2, and Hras1, and overexpression of TEX11 had different effects on their expression. It caused significant increase in the expression of Bax, had little effect on Bcl2, and further suppressed the expression of Hras1. The effects were not affected by PD treatment, suggesting that they were not mediated through estrogen nongenomic pathways. Previous studies have shown that the expressions of c-Fos, Ccnd1, Ccnb1, and Bax could be regulated by estrogen's nongenomic activities (35, 37). Both c-Fos and Ccnd1 contain Sp1-regulatory sequences in their promoters through which the ER can indirectly associate and thus can also be regulated through estrogen's genomic activities. However, our results indicate that in our systems, c-Fos, Ccnd1, and Ccnb1 are regulated only through the nongenomic pathways. Hras1 contains an estrogen response element in its promoter (33), and the observed effects of estrogen is likely medicated through the genomic activity. Similar to cFos and Ccnd1, Bcl2 also contains an Sp1-regulatory sequence in its promoter (36). The lack of effects of TEX11 or PD on the expression of Bcl2 indicates that it is not subjected to estrogen's genomic effects.

Fig. 7.

TEX11 affects the expression of estrogen downstream targets. GC-1 cells transfected with the TEX11 expression vector or the empty vector were treated with or without DPN or PD for 6 h, and the expression levels of the various genes were determined by real-time PCR using Gapdh as an internal control. The results shown represent the averages of three independent experiments. Statistical significance of the differences was determined using the paired t test and is indicated as follows: *, P < 0.05; **, P < 0.01.

In summary, our results showed that TEX11 overexpression reduced the expression of genes such as c-Fos, Ccnd1, Ccnb1, and Hras1 that function in cell proliferation and at the same time increased the expression of the proapoptotic Bax gene.

Discussion

The roles of estrogens in spermatogenesis have been extensively studied, yet the results remain inconclusive (reviewed in Ref. 15). Knocking out Esr1 or Esr2 in mice had no direct effect on spermatogenesis. Male ERα knockout (KO) mice had normal spermatogenesis until about 10 wk of age when the seminiferous tubules started to degenerate due to fluid buildup in the rete testis (38, 39). Male ERβKO mice had normal fertility (40), and the reproductive phenotype of male mice knocked out for both Esr1 and Esr2 was similar to that of ERαKO mice (41). The lack of germ-cell phenotype in ERβKO was unexpected because ERβ is expressed in spermatogonia and SSC. The possibility that GPR30, which as we have shown is also expressed in SSC, provides redundant ER function in the germ cells cannot be ruled out, despite the controversies surrounding it. GPR30 KO mice had normal fertility, similar to ERβKO mice (42). Contrast to ERαKO and ERβKO, deletion of Cyp19, which encodes the key enzyme cytochrome P-450 aromatase in the biosynthesis of estrogens, did impair spermatogenesis (43). Male aromatase KO (ArKO) mice had normal fertility initially but developed impairment between 4.5 months and 1 yr, with spermatogenic arrest at early spermiogenic stages and increased apoptosis. Removing phytoestrogens from the diet worsened the germ cell depletion in ArKO mice, demonstrating that estrogens are important at least in the maintenance of spermatogenesis (44). Whether estrogens also play a role in early male germ cell development during fetal and early postnatal period of mouse development cannot be addressed using the ArKO mice because maternal circulation and milk may provide the needed estrogens. In addition to knocking out genes involved in estrogen signaling, several groups injected E2 or its antagonist ICI 182,780 into wild-type mice or mice with compromised fertility to study their effects on the seminiferous epithelium (15, 45–47). Depending on the system, the effect could be deleterious or beneficial, mediated indirectly through the hypothalamus-pituitary-testes axis or directly on the seminiferous epithelium. Despite the conflicting results obtained from animal studies, a recent study on rat seminiferous tubules cultured in vitro provides strong support for a direct impact of estrogens on germ cell growth (48). E2 and 5α-androstane-3β,17β-diol, which has previously been shown to bind ERβ selectively, stimulated DNA synthesis in spermatogonia, whereas 5α-dihydrotestosterone had no effect. Therefore, there appears to be an ERβ-dependent pathway in spermatogonia that regulates their replication.

In this study, we have linked TEX11 to ERβ through the identification of HPIP as an interacting protein and showed that TEX11 suppressed estrogen-stimulated cell growth. We also showed that Tex11, Hpip, and Esr2 are all expressed in mouse SSC. HPIP is a microtubule binding protein that anchors ERα and ERβ to the cytoskeleton where the proteins form complexes with the Src kinase and PI3K to mediate rapid nongenomic estrogen signaling (20, 21). Our data showed that TEX11 competed with ERβ for the binding to HPIP, promoted ERβ's nuclear translocation and transcription activities, and suppressed its nongenomic activities. Based on these observations, we propose that TEX11 modulates both the genomic and nongenomic actions of ERβ by titrating the amount of ERβ tethered to HPIP. With increased expression of TEX11 in the cells, more ERβ molecules are released from the HPIP complex and free to enter the nucleus to carry out its genomic action. Once ERβ is replaced from the ERβ-HPIP-Src-PI3K complex by TEX11, the complex loses its ability to mediate rapid nongenomic signaling, leading to reduced activation of the AKT and MAPK signaling pathways. The observed effect of TEX11 on E2/DPN-stimulated cell proliferation likely involves both the nongenomic and genomic actions of ERβ. It is well established that the activities of many transcription factors, including the ER, are regulated by phosphorylation (14). Because cell proliferation takes several hours to measure, there is sufficient time for the transcription factors to be phosphorylated through ERβ-mediated signaling pathways and enter the nucleus to act on their target genes. Indeed, we found that TEX11 affected the expression of three known estrogen target genes, c-Fos, Ccnd1, and Ccnb1, through nongenomic effects, whereas it likely affected the expression of Hras1 through genomic effects. It is of great significance that overexpression of TEX11 repressed the expression of several cell proliferation genes and enhanced about 5-fold the expression of the proapoptotic Bax gene. Such a change in gene expression pattern is likely to have a great impact on the growth and survival of the cells.

Our finding that TEX11 suppresses E2-stimulated cell proliferation suggests that increased expression of TEX11 may contribute to the spermatogenic defect associated with KS. Germ-cell degeneration in KS patients occurs during infancy, long before homologous chromosomes pair to form synapses (9). Therefore, it is increased gene expression, not asynapsis of the extra sex chromosome during meiosis (49), that is responsible for the germ cell loss. Due to X-chromosome inactivation, only one of the X-chromosomes in the somatic cells of KS patients is fully active, similar to the situation in normal females. However, about 15% of the genes on the inactive X-chromosome continue to be expressed, albeit at a lower level (50). Thus, the somatic cells of KS patients do express higher levels of these X-inactivation escapees compared with those of normal men. A recent report on the ability of XXY mouse testes to support the development of donor XY germ cells nonetheless suggests that it is the germ cells, not the testis environment, that is defective in KS patients (51). Similar to XX germ cells, the second X-chromosome in XXY germ cells reactivates soon after primordial germ cells enter the genital ridges, leading to twice as high expression of X-linked genes in XXY germ cells as in XY germ cells (52). Because not all genes on the X-chromosome are expressed in germ cells, and increased expression of some but not all genes may be tolerated by the germ cells, it is likely that only a subset of X-linked genes contribute to the germ cell demise in KS patients. The human X-chromosome contains slightly over a thousand genes, and 99 of them are expressed exclusively or predominantly in the testis (3). The KS spermatogenic defect has been mapped to the X-chromosome long arm based on genotype-phenotype correlation in KS patients with aberrant X-chromosomes (53). TEX11 is located on the human X-chromosome long arm. In addition, it is expressed in fetal testes as well as adult testes (5, 54). Its chromosomal location and its expression during early germ cell development make TEX11 a potential candidate responsible for the KS spermatogenic failure. Future generations of transgenic mice carrying an extra copy of Tex11 in a bacteria artificial chromosome transgene may test the actual contribution of TEX11 to KS germ cell death.

Materials and Methods

Animals

The animals used in the study were C57BL/6 mice. They were housed in a specific pathogen-free animal facility, and the experiments had been approved by the Institutional Animal Care and Utilization Committee of Academia Sinica.

Generation and sources of antibodies

A rabbit anti-TEX11 antibody was generated against a synthetic oligopeptide (PPEDQGSVSSTNVAAQNHL) corresponding to the C terminus of the mouse TEX11 and affinity purified using the services of Bethyl Laboratories, Inc. (Montgomery, TX). The sources of the commercial antibodies are as follows: mouse anti-Flag from Sigma (St. Louis, MO), mouse anti-HA from Covance (Emeryville, CA), mouse anti-β-actin from Sigma), mouse anti-AKT from BD Biosciences (Sparks, MD), rabbit anti-p-AKT from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), rabbit anti-ERK1/2 from Biosource (Carlsbad, CA), rabbit anti-p-ERK from Biosource, goat anti-lamin A/C from Santa Cruz, rabbit anti-α-tubulin from Novus (Littleton, CO), rabbit anti-ERα and anti-ERβ from GeneTex (Hsinchu, Taiwan), rabbit anti-GPR125 from Abcam (Cambridge, MA), rabbit anti-EpCAM from Abcam), allophycocyanin (APC)-anti-BrdU from BD Pharmingen (Sparks, MD), fluorescein isothiocyanate-mouse anti-HA from Covance, phycoerythrin-rat anti-CD90.2 (Thy-1.2) from BD Biosciences, Alexa 488-goat antirabbit antibody from Invitrogen (Carlsbad, CA), and Alexa 594-donkey antimouse antibody from Invitrogen.

Plasmid construction

The open reading frames for TEX11 and ERβ were RT-PCR amplified from mouse testis RNA, whereas that for HPIP was amplified from human testis RNA. These cDNA fragments were cloned into pCR2.1-TOPO (Invitrogen) and sequenced. The Tex11 cDNA was then subcloned into pXJ-HA (55) to generate pHA-TEX11, which encodes a full-length TEX11 with an HA epitope at the N terminus. Three additional TEX11 expression constructs encoding truncated TEX11 derivatives, D1 (amino acid residues 1–575), D2 (1–876), and D3 (1–674, 872–947), were generated by PCR amplification of the corresponding coding regions and cloned into pXJ-HA. The HPIP cDNA was cloned into p3xFLAG-CMV-14 (Sigma) to generate pHPIP-Flag, which encodes an HPIP tagged at the C terminus with three copies of the Flag epitope. The ESR2 cDNA was cloned into pcDNA3.1/myc-His (Invitrogen) to generate pERβ-Myc encoding ERβ with a Myc epitope at the C terminus. In addition, the promoter region (from −307 to +58) of the complement component 3 (C3) gene (56) was PCR amplified from human genomic DNA and subcloned into pGL3-Basic (Promega, San Luis Obispo, CA) to generate the firefly luciferase reporter construct pGL3-C3.

Yeast two-hybrid screen

Segments containing the coding regions of the N-terminal (1–393) and the C-terminal (298–947) portions of TEX11 were PCR amplified separately from the Tex11 cDNA clone and inserted into pAS2-1 (Clontech, Palo Alto, CA) to generate the N-terminal and the C-terminal bait plasmids. The bait plasmids were used to screen the human testis MATCHMAKER cDNA library in pACT2 (no. 638810; Clontech) according to the protocols provided by the manufacturer. Briefly, the bait plasmid and target plasmids isolated from the cDNA library were sequentially transformed into yeast reporter strain Y190. Over one million transformants were plated on synthetic defined medium without tryptophan, leucine, and histidine, but with 25 mm 3-aminotriazole (Sigma). Candidate clones that grew on the plates were further tested for the expression of the second reporter gene, lacZ. The lacZ-positive clones were plated on synthetic defined medium containing 10 μg/ml cycloheximide (Sigma) to select for colonies that had lost the bait plasmid. The retained target plasmids were then isolated and sequenced. Interactions between the bait and the candidates were subsequently verified by mating Y190 strains containing the target plasmids with Y187 strains containing the bait vector, the empty vector, or the pLAM5-negative control.

Cell culture and transfection

GC-1, GC-2, and COS7 cells were maintained in DMEM (Hyclone, Fremont, CA) supplemented with 13% fetal bovine serum (FBS), 1× nonessential amino acids, and 1% penicillin/streptomycin (GIBCO, Eggenstein, Germany), whereas MCF7 cells were maintained in RPMI 1640 (GIBCO) supplemented with 10% FBS and 1% penicillin/streptomycin. For transfection and hormone treatment experiments, cells were seeded in six-well plates (2 × 10⁵ per well) or 6-cm dishes (1 × 10⁶ per dish) containing phenol-red-free DMEM or RPMI supplemented with 10% charcoal-stripped FBS (Hyclone) and 1% penicillin/streptomycin. After overnight culturing, the cells were transfected with the various constructs (usually a total of 1 μg DNA per well or 2 μg DNA per dish) using Turbofect (Fermentas, Burlington, Ontario, Canada). The transfection efficiency was around 50% based on the percentages of GFP⁺ cells observed after transfection with a GFP-expression vector.

Coimmunoprecipitation

Twenty-four hours after transfection with the desired constructs, cells in six-well plates were harvested and lysed in 50 μl/well of the Nonidet P-40 (NP-40) lysis buffer [50 mm Tris (pH 8.0), 150 mm NaCl, 0.5% NP-40, and 1× protease inhibitor cocktail). The lysates were mixed with specific antibodies and protein-G Sepharose resin slurry (Amershan, Arlington Heights, IL) to a final concentration of 10% and incubated for 4 h at 4 C. The beads were then washed three times with TBS buffer containing 0.1% Tween 20, and the captured immunocomplexes were eluted by boiling in sodium dodecyl sulfate-sample buffer and analyzed by Western blot using the Immobilon-FL membrane (Millipore, Bedford, MA) and the chemiluminescence system (Millipore).

Purification and gene expression of SSC

SSC were isolated from mouse testes following the protocol established by Kubota et al. (24). Briefly, testes from eight 7- to 8-dpp pups were stripped off the tunica albuginea, treated with deoxyribonuclease I and trypsin, and filtered through a cell strainer to make a single-cell suspension. Phycoerythrin-rat antimouse CD90.2 (Thy-1.2) antibody was then added to the cell suspension. After incubation at 4 C for 30 min, the cells were washed with PBS-S (3% FBS in PBS) and subjected to FACS using the service of the Flow Cytometry Core at Academia Sinica. The CD90.2-positive and -negative fractions were subsequently stained with anti-GPR125 and anti-EpCAM antibodies to access the purity of the cell populations. Total RNA was isolated from the cells using the Trizol reagent (Invitrogen), and cDNA synthesis was performed using the SuperScript III First-Strand Synthesis System (Invitrogen) according to manufacturer's instructions. PCR primers for the various genes are listed in Supplemental Table 1.

Fractionation of the nuclei and the cytoplasm

GC-1 cells in 6-cm plates were cotransfected with 0.5 μg pERβ-Myc and 0.5 μg pHA-TEX11, pXJ-HA, or pHA-D2. Twenty-four hours after transfection, the cells were treated with or without 10 nm E2 (Sigma) for 5 min and harvested. The cytoplasmic and nuclear fractions of the cells were prepared by centrifugation according to a published protocol (57) and adjusted to the same final volume. Equal-volume aliquots of the fractions were then analyzed by Western blot. Lamin A and α-tubulin were used as the nuclear and the cytoplasmic marker, respectively.

Immunofluorescence staining of ERβ in transfected GC-1 cells

GC-1 cells were seeded on coverslips that were placed in a 24-well plate and transfected with 0.5 μg pERβ-Myc and 0.5 μg pHA-TEX11 or pHA-D2. Twenty-four hours after transfection, the cells were treated with or without 1 μm DPN for 10 min and fixed with 4% paraformaldehyde at room temperature for 15 min. After being washed with PBS, the cells were permeabilized by 0.5% Triton X-100 at room temperature for 10 min and blocked with 5 mg/ml BSA for 30 min. Afterward, the cells were incubated with the rabbit anti-TEX11 antibody and mouse anti-Myc antibody (Invitrogen) overnight at 4 C. After three washes with PBS, the cells were incubated with Alexa 488-goat antirabbit antibody and Alexa 594-goat antimouse antibody at room temperature for 2 h and then counterstained with 5 μg/ml Hoechst at room temperature for 10 min. The fluorescence signals were detected by the LSM700 confocal microscope (Carl Ziess MicroImaging Inc., Thornwood, NY), and the levels of ERβ signal in defined areas in the nuclei and the cytoplasm were quantified by use of the MetaMorph Imaging System (Molecular Devices, Inc., Sunnyvale, CA).

Luciferase reporter assay

MCF7 or GC-1 cells in six-well plates were transfected with 0.5 μg pHA-TEX11 (or the deletion constructs), 0.4 μg pGL3-C3, and 0.1 μg pRL-TK per well. The pRL-TK plasmid (Promega) contains a Renilla luciferase gene driven by the HSV-thymidine kinase promoter and was used as an internal control for transfection efficiency. Twenty four hours after transfection, the cells were treated with 10 nm E2, 1 μm DPN, or 100 nm ICI 182,780 (Sigma) alone or in different combinations for 16 h. The cells were then harvested and analyzed for luciferase activity using the Promega dual-luciferase reporter assay system according to the manufacturer's instruction. An shRNA expression clone TRCN0000026-150 (shEsr2) that targets a sequence near the 3′ end of the Esr2 coding region was purchased from the National RNAi Core Facility Platform in Taiwan and used to knock down the expression of endogenous Esr2 in GC-1 cells. GC-1 cells in six-well plates were transfected with 0.3 μg pHA-TEX11 (or pXJ-HA), 0.3 μg shEsr2 (or shluc), 0.3 μg pGL3-C3, and 0.1 μg pRL-TK per well and treated with or without 1 μm DPN for 16 h before being harvested. The cell lysates were analyzed for luciferase activity and Western blotted for ERβ.

Western analysis on AKT/ERK phosphorylation

Twenty four hours after GC-1 cells were transfected with 1.0 μg pHA-D2, 1.0 μg of the empty vector pXJ-HA, or various amounts of pHA-TEX11, the cells were starved for 6 h in media lacking FBS (58) before the media were replaced with new media containing 1 μm DPN. After 10 min, the cells were lysed in the RIPA buffer [120 mm NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate, 1% deoxycholate, 1× protease inhibitor cocktail, and 10 mm Tris (pH 6.8)] and subjected to Western analysis using antibodies specific for the unphosphorylated and phosphorylated AKT/ERK. For experiments involving knockdown of the endogenous ERβ, the cells were transfected with 0.5 μg pHA-TEX11 (or pXJ-HA) and 0.5 μg shEsr2 (or shluc) and processed as described above.

Cell proliferation assays

The APC BrdU Flow Kit from BD Pharmingen was used for BrdU labeling of cells. GC-1 or GC-2 cells in three sets of four 6-cm petri dishes were transfected with 2 μg/dish of pXJ-HA, pHA-TEX11, and pHA-D2, respectively, and harvested 5 h after the addition of DNA. Cells transfected with the same vector were pooled and divided into four equal portions and incubated in a CO₂ incubator in phenol-red-free DMEM containing 10% charcoal-stripped FBS and 10 μm BrdU with or without 10 nm E2, 1 μm DPN (Tocris Bioscience, Ellisville, MO), or 10 μm PD (Sigma) in four different combinations. After 10 h, the cells were harvested and a small aliquot was used to determine the total cell counts. The remaining cells were prepared for flow cytometry following the manual included in the BrdU Flow Kit. BrdU and TEX11 (or D2) were detected by the APC-anti-BrdU antibody and the fluorescein isothiocyanate-anti-HA antibody, respectively, and the cells were sorted on a BD FACSCalibur flow cytometer (BD Biosciences) and analyzed using the Cellquest software.

Real-time PCR quantification of gene expression

GC-1 cells cultured in 6-cm petri dishes were transfected with 2 μg/dish of pXJ-HA or pHA-TEX11. Five hours after the addition of DNA, the medium was replaced with phenol-red-free DMEM containing 10% charcoal-stripped FBS, and the cells were incubated in a CO₂ incubator for 16 h. Then the cells were treated with or without 1 μm DPN or 10 μm PD for 6 h before being harvested for RNA isolation. PCR primers for mouse c-Fos (37), Ccnd1 (37), Ccnb1 (59), Bax (60), Bcl-2 (60), and Hras (61) have been published, and Gapdh (5′-ATGTGCCCGTCGTGGATCTG-3′ and 5′-CCTCAGTGTAGCCCAAGATG-3′) served as an internal control. Real-time PCR of these genes were performed with Power SYBR Green (Applied Biosystems, Foster City, CA) in an ABI PRISM 7500 Sequence Detection System (Applied Biosystems). All measurements were carried out in triplicate.