Protein Arginine Methyltransferase 1 Is Essential for the Meiosis of Male Germ Cells.

Protein arginine methyltransferase 1 (PRMT1) is a major enzyme responsible for the formation of methylarginine in mammalian cells; however, its function in vivo is not well understood due to its early embryonic lethality in null mice exhibiting spontaneous DNA damage, cell cycle delays, and defects in check point activation. Here, we generated germ cell-specific Prmt1 knock-out (KO) mice to evaluate the function of PRMT1 in spermatogenesis. Our findings demonstrate that PRMT1 is vital for male fertility in mice. Spermatogenesis in Prmt1 KO mice was arrested at the zygotene-like stage of the first meiotic division due to an elevated number of DNA double-strand breaks (DSBs). There was a loss of methylation in meiotic recombination 11 (MRE11), the key endonuclease in MRE11/RAD50/NBS 1 (MRN) complex, resulting in the accumulation of SPO11 protein in DSBs. The ATM-mediated negative feedback control over SPO11 was lost and, consequently, the repair pathway of DSBs was highly affected in PRMT1 deficient male germ cells. Our findings provide a novel insight into the role of PRMT1-mediated asymmetric demethylation in mouse spermatogenesis.

NOTCH1 intracellular domain negatively regulates PAK1 signaling pathway through direct interaction.

p21-Activated kinase 1 (PAK1) is a serine/threonine protein kinase implicated in cytoskeletal remodeling and cell motility. Recent studies have shown that it also promotes cell proliferation, regulates apoptosis, and increases cell transformation and invasion. In this study, we showed that NOTCH1 intracellular domain (NOTCH1-IC) negatively regulated PAK1 signaling pathway. We found a novel interaction between NOTCH1-IC and PAK1. Overexpression of NOTCH1-IC decreased PAK1-induced integrin-linked kinase 1 (ILK1) phosphorylation, whereas inhibition of NOTCH1 signaling increased PAK1-induced ILK1 phosphorylation. Notably, ILK1 phosphorylation was higher in PS1,2(-/-) cells than in PS1,2(+/+) cells. As expected, overexpression of NOTCH1-IC decreased ILK1-induced phosphorylation of glycogen synthase kinase 3 beta (GSK-3beta). Furthermore, NOTCH1-IC disrupted the interaction of PAK1 with ILK1 and altered PAK1 localization by directly interacting with it. This inhibitory effect of NOTCH1-IC on the PAK1 signaling pathway was mediated by the binding of NOTCH1-IC to PAK1 and by the alteration of PAK1 localization. Together, these results suggest that NOTCH1-IC is a new regulator of the PAK1 signaling pathway that directly interacts with PAK1 and regulates its shuttling between the nucleus and the cytoplasm.

Testis-Specific GTPase (TSG): An oligomeric protein.

BACKGROUND: Ras-related proteins in brain (Rab)-family proteins are key members of the membrane trafficking pathway in cells. In addition, these proteins have been identified to have diverse functions such as cross-talking with different kinases and playing a role in cellular signaling. However, only a few Rab proteins have been found to have a role in male germ cell development. The most notable functions of this process are performed by numerous testis-specific and/or germ cell-specific genes. Here, we describe a new Rab protein that is specifically expressed in male germ cells, having GTPase activity. RESULTS: Testis-specific GTPase (TSG) is a male-specific protein that is highly expressed in the testis. It has an ORF of 1593 base pairs encoding a protein of 530 amino acids. This protein appears in testicular cells approximately 24 days postpartum and is maintained thereafter. Immunohistochemistry of testicular sections indicates localized expression in germ cells, particularly elongating spermatids. TSG has a bipartite nuclear localization signal that targets the protein to the nucleus. The C-terminal region of TSG contains the characteristic domain of small Rab GTPases, which imparts GTPase activity. At the N-terminal region, it has a coiled-coil motif that confers self-interaction properties to the protein and allows it to appear as an oligomer in the testis. CONCLUSION: TSG, being expressed in the male gonad in a developmental stage-specific manner, may have a role in male germ cell development. Further investigation of TSG function in vivo may provide new clues for uncovering the secrets of spermatogenesis.

NUP50 is necessary for the survival of primordial germ cells in mouse embryos.

Nucleoporin 50 kDa (NUP50), a component of the nuclear pore complex, is highly expressed in male germ cells, but its role in germ cells is largely unknown. In this study, we analyzed the expression and function of NUP50 during the embryonic development of germ cells using NUP50-deficient mice. NUP50 was expressed in germ cells of both sexes at embryonic day 15.5 (E15.5), E13.5, and E12.5. In addition, NUP50 expression was also detected in primordial germ cells (PGCs) migrating into the genital ridges at E9.5. The gonads of Nup50-/- embryos of both sexes contained few PGCs at both E11.5 and E12.5 and no developing germ cells at E15.5. The migratory PGCs in Nup50-/- embryos at E9.5 showed increased apoptosis but a normal rate of proliferation, resulting in the progressive loss of germ cells at later stages. Taken together, these results suggest that NUP50 plays an essential role in the survival of PGCs during embryonic development.

Puberty-related changes in cortisol, dehydroepiandrosterone, and estradiol-17ß secretions within the first hour after waking in premenarcheal girls.

OBJECTIVE: The onset of menstruation is the hallmark of female pubertal development. The present study determined whether pubertal girls experience adrenocortical and ovarian steroid secretions within their first waking hour before getting their period, similar to those observed in adult females with regular cycles. METHODS: Cortisol, dehydroepiandrosterone (DHEA), and estradiol-17ß concentrations were measured in saliva samples collected after awakening (0, 30, and 60 min after awakening) from 158 normal premenarcheal pubertal girls and 69 adult females with regular menstrual cycles. The girls were subgrouped according to self-reported Tanner breast (B) and pubic hair (PH) stages (B1PH1, B2PH1, B2PH2, B3PH1, and B3PH2). RESULTS: All the subgroups showed a similar pattern of cortisol secretion. However, cortisol levels were higher in girls at B3PH1 and at B3PH2 than other subgroups. DHEA secretion showed a similar pattern across the groups examined. The largest increase in DHEA levels occurred between B1PH1 and B2PH1 stages, and further increased with pubertal progression. DHEA levels in girls at B3PH2 were approximately one half of the adult value. Estradiol-17ß profiles in girls at B3PH1 and B3PH2 differed from those of other subgroups of girl. A sharp increase in estradiol-17ß levels after awakening which observed in adult females emerged in girls at B3PH1 and B3PH2. However, the estradiol-17ß levels did not reach adult values until B3PH2 stage. CONCLUSIONS: The progression of female puberty includes an increase in the levels of adrenocortical and ovarian steroid secretions and a gain of adult female-like patterns of estradiol-17ß secretion within their first waking hour.

The LKB1-TSSK1B axis controls YAP phosphorylation to regulate the Hippo-YAP pathway.

The Hippo pathway's main effector, Yes-associated protein (YAP), plays a crucial role in tumorigenesis as a transcriptional coactivator. YAP's phosphorylation by core upstream components of the Hippo pathway, such as mammalian Ste20 kinase 1/2 (MST1/2), mitogen-activated protein kinase kinase kinase kinases (MAP4Ks), and their substrate, large tumor suppressor 1/2 (LATS1/2), influences YAP's subcellular localization, stability, and transcriptional activity. However, recent research suggests the existence of alternative pathways that phosphorylate YAP, independent of these core upstream Hippo pathway components, raising questions about additional means to inactivate YAP. In this study, we present evidence demonstrating that TSSK1B, a calcium/calmodulin-dependent protein kinase (CAMK) superfamily member, is a negative regulator of YAP, suppressing cellular proliferation and oncogenic transformation. Mechanistically, TSSK1B inhibits YAP through two distinct pathways. Firstly, the LKB1-TSSK1B axis directly phosphorylates YAP at Ser94, inhibiting the YAP-TEAD complex's formation and suppressing its target genes' expression. Secondly, the TSSK1B-LATS1/2 axis inhibits YAP via phosphorylation at Ser127. Our findings reveal the involvement of TSSK1B-mediated molecular mechanisms in the Hippo-YAP pathway, emphasizing the importance of multilevel regulation in critical cellular decision-making processes.

Structure-based virtual screening and identification of a novel androgen receptor antagonist.

Hormonal therapies, mainly combinations of anti-androgens and androgen deprivation, have been the mainstay treatment for advanced prostate cancer because the androgen-androgen receptor (AR) system plays a pivotal role in the development and progression of prostate cancers. However, the emergence of androgen resistance, largely due to inefficient anti-hormone action, limits the therapeutic usefulness of these therapies. Here, we report that 6-(3,4-dihydro-1H-isoquinolin-2-yl)-N-(6-methylpyridin-2-yl)nicotinamide (DIMN) acts as a novel anti-androgenic compound that may be effective in the treatment of both androgen-dependent and androgen-independent prostate cancers. Through AR structure-based virtual screening using the FlexX docking model, fifty-four compounds were selected and further screened for AR antagonism via cell-based tests. One compound, DIMN, showed an antagonistic effect specific to AR with comparable potency to that of the classical AR antagonists, hydroxyflutamide and bicalutamide. Consistent with their anti-androgenic activity, DIMN inhibited the growth of androgen-dependent LNCaP prostate cancer cells. Interestingly, the compound also suppressed the growth of androgen-independent C4-2 and CWR22rv prostate cancer cells, which express a functional AR, but did not suppress the growth of the AR-negative prostate cancer cells PPC-1, DU145, and R3327-AT3.1. Taken together, the results suggest that the synthetic compound DIMN is a novel anti-androgen and strong candidate for useful therapeutic agent against early stage to advanced prostate cancer.

Phosphoenolpyruvate carboxykinase and glucose-6-phosphatase are required for steroidogenesis in testicular Leydig cells.

Cyclic AMP (cAMP) induces steroidogenic enzyme gene expression and stimulates testosterone production in Leydig cells. Phosphoenolpyruvate carboxykinase (PEPCK) is expressed in Leydig cells, but its role has not been defined. In this study, we found that PEPCK and glucose-6-phosphatase (Glc-6-Pase) are increased significantly following cAMP treatment of mouse Leydig cells. Moreover, cAMP treatment increased recruitment of the cAMP-response element-binding transcription factor and decreased recruitment of the corepressor DAX-1 on the pepck promoter. Furthermore, cAMP induced an increase in ATP that correlated with a decrease in phospho-AMP-activated protein kinase (AMPK). In contrast, knockdown or inhibition of PEPCK decreased ATP and increased phospho-AMPK. Treatment with an AMPK activator or overexpression of the constitutively active form of AMPK inhibited cAMP-induced steroidogenic enzyme promoter activities and gene expression. Liver receptor homolog-1 (LRH-1) was involved in cAMP-induced steroidogenic enzyme gene expression but was inhibited by AMPK activation in Leydig cells. Additionally, inhibition or knockdown of PEPCK and Glc-6-Pase decreased cAMP-mediated induction of steroidogenic enzyme gene expression and steroidogenesis. Finally, pubertal mouse (8-week-old) testes and human chorionic gonadotropin-induced prepubertal mouse testes showed increased PEPCK and Glc-6-Pase gene expression. Taken together, these results suggest that induction of PEPCK and Glc-6-Pase by cAMP plays an important role in Leydig cell steroidogenesis.

Testicular steroidogenesis is locally regulated by androgen via suppression of Nur77.

Steroidogenesis in the testis is regulated by a negative feedback mechanism through the hypothalamus-pituitary-testis axis. Recent studies suggest that besides this long-loop regulation, testicular steroidogenesis is also locally regulated by androgen. However, the molecular mechanism behind this additional regulatory pathway has been poorly addressed. In the present study, we demonstrate that liganded androgen receptor (AR) suppresses the transcriptional activity of Nur77 on steroidogenic enzyme gene promoters, affecting testicular steroidogenesis. AR physically interacts and colocalizes with Nur77 in the nucleus in the presence of androgen. AR inhibits Nur77 transactivation by competing mainly with coactivators such as SRC-1 for Nur77 binding. These results suggest that androgen, through binding to AR, directly acts as a signal inhibiting the expression of steroidogenic enzyme genes in Leydig cells, eventually resulting in decreased testicular steroidogenesis. These findings strongly support the hypothesis that androgen acts locally to regulate testicular steroidogenesis, and may provide its action mechanism.

Suppression of estrogen receptor-alpha transactivation by thyroid transcription factor-2 in breast cancer cells.

Estrogen receptors (ERs), which mediate estrogen actions, regulate cell growth and differentiation of a variety of normal tissues and hormone-responsive tumors through interaction with cellular factors. In this study, we show that thyroid transcription factor-2 (TTF-2) is expressed in mammary gland and acts as ERα co-repressor. TTF-2 inhibited ERα transactivation in a dose-dependent manner in MCF-7 breast cancer cells. In addition, TTF-2 directly bound to and formed a complex with ERα, colocalizing with ERα in the nucleus. In MCF-7/TTF-2 stable cell lines, TTF-2 repressed the expression of endogenous ERα target genes such as pS2 and cyclin D1 by interrupting ERα binding to target promoters and also significantly decreased cell proliferation. Taken together, these data suggest that TTF-2 may modulate the function of ERα as a corepressor and play a role in ER-dependent proliferation of mammary cells.

TR3 Enhances AR Variant Production and Transactivation, Promoting Androgen Independence of Prostate Cancer Cells.

The pro-oncogenic function of TR3, an orphan nuclear receptor, has been reported in prostate cancer. However, the roles of TR3 in androgen receptor (AR) expression and signaling in prostate cancer cells are poorly understood. Database analysis revealed that TR3 expression level is elevated in prostate tumors, and is positively, although weakly, correlated with that of AR. TR3 overexpression increased the production of AR splice variants in addition to general upregulation of AR expression. TR3 interacted with some spliceosomal complex components and AR precursor mRNA, altering the splice junction rates between exons. TR3 also enhanced androgen-independent AR function. Furthermore, TR3 overexpression increased cell proliferation and mobility of AR-positive prostate cancer cells and stimulated tumorigenesis of androgen-independent prostate cancer cells in mouse xenograft models. This is the first study to report that TR3 is a multifunctional regulator of AR signaling in prostate cancer cells. TR3 alters AR expression, splicing process, and activity in prostate cancer cells, increasing the androgen independence of AR signaling. Therefore, TR3 may play a crucial role in the progression of prostate cancer to an advanced castration-resistant form.

JAG1 Intracellular Domain Enhances AR Expression and Signaling and Promotes Stem-like Properties in Prostate Cancer Cells.

JAG1 expression is upregulated in high-grade metastatic prostate carcinomas and associated with poor disease-free survival of patients with prostate cancer. Intriguingly, all JAG1-positive prostate carcinomas express JICD although JICD function in prostate cancer (PC) cells is poorly understood. In this study, we found that JICD overexpression increased the expression levels of AR, especially AR-Vs, in PC cell lines and significantly enhanced androgen-independent and androgen-dependent function of ARs. Interestingly, JICD overexpression upregulated the expression of the PCSC marker CD133 in PC cells as the expression of self-renewal markers; namely, NANOG and OCT3/4 increased. In addition, JICD overexpression highly increased the expression of anti-apoptotic BCL-XL protein, while it little affected the expression of apoptotic BIM protein. In 3D cell culture assays, the spheres formed by JICD-overexpressing PC subline cells (C4-2 and CWR22Rv1) were larger than those formed by control (EV) subline cells with undifferentiated morphology. Although JICD overexpression caused quiescence in cell proliferation, it activated the expression of components in PCSC-related signaling pathways, increased PC cell mobility, and promoted in vivo xenograft mouse tumorigenesis. Therefore, JICD may play a crucial role in enhancing androgen independence and promoting stem-like properties in PC cells and should be considered a novel target for CRPC and PCSC diagnostic therapy.

Leydig Cell-Specific DAX1-Deleted Mice Has Higher Testosterone Level in the Testis During Pubertal Development.

Testosterone, the male sex hormone, is necessary for the development and function of the male reproductive system. Biosynthesis of testosterone in mammals mainly occurs in testicular Leydig cells. Many proteins such as P450c17, 3ß-HSD, and StAR are involved in testicular steroidogenesis. DAX1 is essential for sex development and interacts with nuclear receptors such as steroidogenic factor 1 to inhibit steroidogenesis. In this study, we investigated the role of DAX1 in testicular steroidogenesis in vivo by generating Leydig cell-specific DAX1-knockout mice. Radioimmunoassay revealed that the levels of testosterone and progesterone were higher in Leydig cell-specific DAX1-knockout testes than in the testes from wild-type mice during the first 3-4 weeks of aging. In addition, the expression levels of steroidogenic genes, such as StAR, P450c17, P450scc, and 3ß-HSD, were considerably higher in the testes from DAX1-knockout mice. DAX1-deficient mouse testes seemed to attain early puberty with the acceleration of germ cell development. These data suggest that DAX1 regulates the expression of steroidogenic genes, and thereby controls and fine-tunes steroidogenesis during testis development.

Anti-steroidogenic factor ARR19 inhibits testicular steroidogenesis through the suppression of Nur77 transactivation.

ARR19 (androgen receptor corepressor-19 kDa), a leucine-rich protein whose expression is down-regulated by luteinizing hormone and cAMP, is differentially expressed during the development of Leydig cells and inhibits testicular steroidogenesis by reducing the expression of steroidogenic enzymes. However, the molecular events behind the suppression of testicular steroidogenesis are unknown. In the present study, we demonstrate that ARR19 inhibits the transactivation of orphan nuclear receptor Nur77, which is one of the major transcription factors that regulate the expression of steroidogenic enzyme genes in Leydig cells. ARR19 physically interacts with Nur77 and suppresses Nur77-induced promoter activity of steroidogenic enzyme genes including StAR, P450c17, and 3beta-HSD in Leydig cells. Transient transfection and chromatin immunoprecipitation assays revealed that ARR19-mediated reduced expression of steroidogenic enzyme genes was likely due to the interference of SRC-1 recruitment to Nur77 protein on the promoter of steroidogenic enzyme genes. These findings suggest that ARR19 acts as a novel coregulator of Nur77, in turn regulating Nur77-induced testicular steroidogenesis, and may play an important role in the development and function of testicular Leydig cells.

FOXA3, a Negative Regulator of Nur77 Expression and Activity in Testicular Steroidogenesis.

Biosynthesis of testosterone occurs mainly in the testicular Leydig cells. Nur77, an orphan nuclear receptor that is expressed in response to the luteinizing hormone/cyclic adenosine monophosphate (LH/cAMP) signaling pathway, is one of the key factors that regulate steroidogenesis in Leydig cells. The function of Nur77 is modulated through interaction with other proteins. FOXA3, a transcription factor that is crucial for male fertility, is also expressed in Leydig cells. Here, we sought to elucidate the role of FOXA3 in testicular steroidogenesis by focusing on its interaction with Nur77. LH/cAMP signaling induces the onset of steroidogenesis in Leydig cells but has a repressive effect on the expression of FOXA3. Overexpression of FOXA3 in MA-10 Leydig cells repressed cAMP-induced expression of Nur77 and its target steroidogenic genes (StAR, P450c17, and Hsd3ß). Furthermore, FOXA3 suppressed Nur77 transactivation of the promoter of steroidogenic genes. In mouse primary Leydig cells, adenovirus-mediated overexpression of FOXA3 had similar effects and resulted in decreased production of testosterone. Taken together, these results suggest the role of FOXA3 in the regulation of steroidogenic genes in Leydig cells and fine-tuning steroidogenesis in the testis.

ARR19 (androgen receptor corepressor of 19 kDa), an antisteroidogenic factor, is regulated by GATA-1 in testicular Leydig cells.

ARR19 (androgen receptor corepressor of 19 kDa), which encodes for a leucine-rich protein, is expressed abundantly in the testis. Further analyses revealed that ARR19 was expressed in Leydig cells, and its expression was differentially regulated during Leydig cell development. Adenovirus-mediated overexpression of ARR19 in Leydig cells inhibited testicular steroidogenesis, down-regulating the expression of steroidogenic enzymes, which suggests that ARR19 is an antisteroidogenic factor. Interestingly, cAMP/luteinizing hormone attenuated ARR19 expression in a fashion similar to that of GATA-1, which was previously reported to be down-regulated by cAMP. Sequence analysis of the Arr19 promoter revealed the presence of two putative GATA-1 binding motifs. Further analyses with 5' deletion and point mutants of putative GATA-1 binding motifs showed that these GATA-1 binding sites were critical for high promoter activity. CREB-binding protein coactivated GATA-1 and markedly increased the activity of the Arr19 promoter. Both GATA-1 and CREB-binding proteins occupied the GATA-1 motifs within the Arr19 promoter, which was repressed by cAMP treatment. Altogether, these findings demonstrate that ARR19 is the target gene of GATA-1 and suggest that ARR19 gene expression in testicular Leydig cells is regulated by luteinizing hormone/cAMP signaling via the control of GATA-1 expression, resulting in the control of testicular steroidogenesis.

Hakai acts as a coregulator of estrogen receptor alpha in breast cancer cells.

Estrogen receptors play a key role in breast cancer development. One of the current therapeutic strategies for the treatment of estrogen receptor (ER)-α-positive breast cancers relies on the blockade of ERα transcriptional activity. In the present study, we characterized Hakai, originally characterized as an E-cadherin binding protein, as a strong blockade of ERα in breast cancer cells. We showed that Hakai inhibited the transcriptional activity of ERα by binding directly to ERα. The DNA-binding domain of ERα was found to be responsible for its interaction with Hakai. Hakai competed with ERα coactivators, such as steroid receptor coactivator-1 (SRC-1) and glucocoriticord receptor interacting protein-1 (GRIP-1), for the modulation of ERα transactivation, while its ubiquitin-ligase activity was not required. Further, overexpression of Hakai inhibited the proliferation and migration of breast cancer cells. Taken together, these results suggest that Hakai is a novel corepressor of ERα and may play a negative role in the development and progression of breast cancers.

Overexpression of hepatocyte nuclear factor-3alpha induces apoptosis through the upregulation and accumulation of cytoplasmic p53 in prostate cancer cells.

BACKGROUND: Hepatocyte nuclear factor-3alpha (HNF-3alpha) has been known to act as a repressor in the pathogenesis of many cancers. Herein, we investigated the effect of HNF-3alpha overexpression in prostate cancer cells. METHODS: HNF-3alpha was overexpressed in prostate cancer cells using an adenovirus recombinant expressing wild-type HNF-3alpha. The apoptosis of prostate cancer cells was determined by TUNEL, FACS, and caspase activity analyses. RESULTS: Adenovirus-mediated overexpression of HNF-3alpha caused cell death in prostate cancer cells as assessed by changes in cellular and nuclear morphology, TUNEL analysis, and caspase activations. Furthermore, FACS analysis showed an increased sub-G1 phase of cell cycle as well as the G2/M phase with a corresponding decrease in S phases. HNF-3alpha overexpression caused the upregulation of p53 protein and its accumulation, together with HNF-3alpha, in the cytoplasm. It also causes Bax protein to localize to the mitochondria-enriched fraction. These findings suggest that multiple apoptotic pathways seem to be involved in the HNF-3alpha-induced cell death: pathways involving the accumulation of p53 protein in the cytoplasm and subsequent cytochrome c release, and other pathways involving death receptor signaling and caspase-8 activation. CONCLUSIONS: The results of the current study suggest a novel function of HNF-3alpha as a killer of malignant prostate cancer cells, which reveals HNF-3alpha as a promising therapeutic molecule for prostate cancers.

Testicular steroidogenesis is decreased by hyperthermia in old rats.

PURPOSE: To evaluate the effects of hyperthermia on testicular steroidogenesis in a rat model. MATERIALS AND METHODS: Three-month-old and 20-month-old male Sprague-Dawley rats were randomly divided into 4 groups of 10 rats each, a control group and a hot-bath group for each age. The rats in the hot-bath groups received multiple 10-min treatments in a hot bath (41-43 degrees C) over a period of 4 weeks. Testicular testosterone, serum testosterone and serum luteinizing hormone levels were measured. The protein levels of 2 steroidogenic enzymes, StAR and P450c17, were measured by Western blot. The testes were examined histologically by light microscopy. RESULTS: Testicular testosterone levels of the 20-month-old, but not the 3-month-old, rats in the hot-bath group were significantly lower than those in the control group (p < 0.05). Serum testosterone levels of both the old and the young hot-bath groups tended to decrease compared with their corresponding controls, although the differences were not statistically significant. Serum luteinizing hormone levels changed insignificantly after the hot baths in both age groups. The hot-bath treatment had no significant effect on P450c17 protein levels, whereas the protein level of StAR was significantly lower in the old hot-bath group than in the same-age control group (p < 0.05). CONCLUSIONS: Hyperthermia significantly decreased the testicular testosterone level in old male rats and significantly lowered the StAR protein level. These data imply that frequent hot baths might impair testicular steroidogenesis, especially in old men.