Dosage-sensitive Sex Reversal-adrenal Hypoplasia Research Articles

Identification and Functional Evaluation of Alternative Splice Variants of Dax1 in Mouse Embryonic Stem Cells.

Dax1 (Nr0b1; Dosage-sensitive sex reversal-adrenal hypoplasia congenital on the X-chromosome gene-1) is an important component of the transcription factor network that governs pluripotency in mouse embryonic stem cells (ESCs). Functional evaluation of alternative splice variants of pluripotent transcription factors has shed additional insight on the maintenance of ESC pluripotency and self-renewal. Dax1 splice variants have not been identified and characterized in mouse ESCs. We identified 18 new transcripts of Dax1 with putative protein-coding properties and compared their protein structures with known Dax1 protein (Dax1-472). The expression pattern analysis showed that the novel isoforms were cotranscribed with Dax1-472 in mouse ESCs, but they had transcriptional heterogeneity among single cells and the subcellular localization of the encoded proteins differed. Cell function experiments indicated that Dax1-404 repressed Gata6 transcription and functionally replaced Dax1-472, while Dax1-38 and Dax1-225 partially antagonized Dax1-472 transcriptional repression. This study provided a comprehensive characterization of the Dax1 splice variants in mouse ESCs and suggested complex effects of Dax1 variants in a self-renewal regulatory network.

Inhibition of GLI Transcriptional Activity and Prostate Cancer Cell Growth and Proliferation by DAX1.

The Hedgehog (Hh) signaling pathway plays an essential role in the initiation and progression of prostate cancer. This is mediated by transcriptional factors belonging to the GLI (glioma-associated oncogene) family, which regulate downstream targets to drive prostate cancer progression. The activity of GLI proteins is tightly controlled by a range of mechanisms, including molecular interactions and post-translational modifications. In particular, mitogenic and oncogenic signaling pathways have been shown to regulate GLI protein activity independently of upstream Hh pathway signaling. Identifying GLI protein regulators is critical for the development of targeted therapies that can improve patient outcomes. This study aimed to identify a novel protein that directly regulates the activity of GLI transcription factors in prostate cancer. We performed gene expression, cellular analyses, and reporter assays to demonstrate that DAX1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) interacts with GLI1 and GLI2, the master regulators of Hh signaling. Interestingly, DAX1 overexpression significantly inhibited Hh signaling by reducing GLI1 and GLI2 activity, prostate cancer cell proliferation, and viability. Our results shed light on a novel regulatory mechanism of Hh signaling in prostate cancer cells. The interaction between DAX1 and GLI transcription factors provides insight into the complex regulation of Hh signaling in prostate cancer. Given the importance of Hh signaling in prostate cancer progression, targeting DAX1-GLI interactions may represent a promising therapeutic approach against prostate cancer. Overall, this study provides new insights into the regulation of the Hh pathway and its role in prostate cancer progression. The findings suggest that DAX1 could serve as a potential therapeutic target for the treatment of prostate cancer.

The Expression Pattern of Insulin-Like Growth Factor Subtype 3 (igf3) in the Orange-Spotted Grouper Epinephelus coioides and Its Function on Ovary Maturation.

A new insulin-like growth factor (Igf) subtype 3 (igf3) has recently been found in the bony fish orange-spotted grouper (Epinephelus coioides). However, the role of igf3 in the maturation of the ovary and sex differentiation in E. coioides is currently unknown. We examined the ovarian localization and receptor binding of the novel ortholog Igf3 using qRT-PCR, and Western blotting, combined with in situ hybridization and immunohistochemistry methods. Results demonstrated the presence of igf3 mRNA and protein in mature oocytes. Furthermore, Igf3 protein expression was not detected in testis, brain, kidney and liver homogenates. The calculated molecular weight of Igf3 was 22 kDa, which was consistent with the deduced amino acid sequence from the full-length open reading frame. The immunoreactivity showed that Igf3 was strongly present in the follicle staining fully-grown stage. The igf3 mRNA expression level was significantly positively correlated with ovarian follicular maturation. Meanwhile, Igf3 increased germinal-vesicle breakdown in a time- and dose-dependent manner. In vitro, treatment of primary ovarian cells with Igf3 up-regulated significantly the mRNA expression level of genes related to sex determination and reproduction such as forkhead boxl2 (foxl2), dosage-sensitive sex reversal adrenal hypoplasia critical region on chromosome x gene 1 (dax1), cytochrome P450 family 19 subfamily member 1 a (cyp19a1a), cytochrome P450 family 11 subfamily a member 1 a (cyp11a1a) and luteinizing hormone receptor 1 (lhr1). Overall, our results demonstrated that igf3 promotes the maturation of the ovary and plays an important role in sex differentiation in E. coioides.

Hepatocyte DAX1 Deletion Exacerbates Inflammatory Liver Injury by Inducing the Recruitment of CD4+ and CD8+ T Cells through NF-κB p65 Signaling Pathway in Mice.

Fulminant hepatitis is characterized by rapid and massive immune-mediated liver injury. Dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1; NR0B1) represses the transcription of various genes. Here, we determine whether DAX1 serves as a regulator of inflammatory liver injury induced by concanavalin A (ConA). C57BL/6J (WT), myeloid cell-specific Dax1 knockout (MKO), and hepatocyte-specific Dax1 knockout (LKO) mice received single intravenous administration of ConA. Histopathological changes in liver and plasma alanine aminotransferase and aspartate aminotransferase levels in Dax1 MKO mice were comparable with those in WT mice following ConA administration. Unlike Dax1 MKO mice, Dax1 LKO mice were greatly susceptible to ConA-induced liver injury, which was accompanied by enhanced infiltration of immune cells, particularly CD4+ and CD8+ T cells, in the liver. Factors related to T-cell recruitment, including chemokines and adhesion molecules, significantly increased following enhanced and prolonged phosphorylation of NF-κB p65 in the liver of ConA-administered Dax1 LKO mice. This is the first study to demonstrate that hepatocyte-specific DAX1 deficiency exacerbates inflammatory liver injury via NF-κB p65 activation, thereby causing T-cell infiltration by modulating inflammatory chemokines and adhesion molecules. Our results suggest DAX1 as a therapeutic target for fulminant hepatitis treatment.

Hepatocyte-Specific Deficiency of DAX-1 Protects Mice from Acetaminophen-Induced Hepatotoxicity by Activating NRF2 Signaling.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, but its overdose can cause acute liver failure. The dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1, NR0B1), is an orphan nuclear receptor that acts as a transcriptional co-repressor of various genes. In this study, we identified the role of DAX-1 in APAP-induced liver injury using hepatocyte-specific Dax-1 knockout (Dax-1 LKO) mice. Mouse primary hepatocytes were used as a comparative in vitro study. APAP overdose led to decreased plasma alanine aminotransferase and aspartate aminotransferase levels in Dax-1 LKO mice compared to C57BL/6J (WT) controls, accompanied by reduced liver necrosis. The expression of the genes encoding the enzymes catalyzing glutathione (GSH) synthesis and metabolism and antioxidant enzymes was increased in the livers of APAP-treated Dax-1 LKO mice. The rapid recovery of GSH levels in the mitochondrial fraction of APAP-treated Dax-1 LKO mice led to reduced reactive oxygen species levels, resulting in the inhibition of the prolonged JNK activation. The hepatocyte-specific DAX-1 deficiency increased the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2) compared with WT controls after APAP administration. These results indicate that DAX-1 deficiency in hepatocytes protects against APAP-induced liver injury by Nrf2-regulated antioxidant defense.

A Novel Muscle Atrophy Mechanism: Myocyte Degeneration Due to Intracellular Iron Deprivation

Muscle atrophy is defined as the progressive degeneration or shrinkage of myocytes and is triggered by factors such as aging, cancer, injury, inflammation, and immobilization. Considering the total amount of body iron stores and its crucial role in skeletal muscle, myocytes may have their own iron regulation mechanism. Although the detrimental effects of iron overload or iron deficiency on muscle function have been studied, the molecular mechanism of iron-dependent muscle atrophy has not been elucidated. Using human muscle tissues and in the mouse rotator cuff tear model, we confirmed an association between injury-induced iron depletion in myocytes and muscle atrophy. In differentiated C2C12 myotubes, the effects of iron deficiency on myocytes and the molecular mechanism of muscle atrophy by iron deficiency were evaluated. Our study revealed that the lower iron concentration in injured muscle was associated with the upregulation of ferroportin, an iron exporter that transports iron out of cells. Ferroportin expression was increased by hypoxia-inducible factor 1α (HIF1α), which is activated by muscle injury, and its expression is controlled by HIF1 inhibitor treatment. Iron deprivation caused myocyte loss and a marked depletion of mitochondrial membrane potential leading to muscle atrophy, together with increased levels of myostatin, the upstream regulator of atrogin1 and muscle RING-finger protein-1 (MuRF1). Myostatin expression under iron deficiency was mediated by an orphan nuclear receptor, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome (DAX1).

Targeting 17β-estradiol biosynthesis in neural stem cells improves stroke outcome.

Dax-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital region on X-chromosome gene 1) blocks 17β-estradiol biosynthesis and its knockdown would be expected to increase 17β-estradiol production. We hypothesized that knockdown of Dax-1 in a conditionally immortalized neural stem cell (NSC) line, MHP36, is a useful approach to increase 17β-estradiol production. Short hairpin (sh) RNA targeted to Dax-1 in NSCs, namely MHP36-Dax1KD cells, resulted in the degradation of Dax-1 RNA and attenuation of Dax-1 protein expression. In vitro, MHP36-Dax1KD cells exhibited overexpression of aromatase and increased 17β-estradiol secretion compared to MHP36 cells. As 17β-estradiol has been shown to promote the efficacy of cell therapy, we interrogated the application of 17β-estradiol-enriched NSCs in a relevant in vivo disease model. We hypothesized that MHP36-Dax1KD cells will enhance functional recovery after transplantation in a stroke model. C57BL/6 male adult mice underwent ischemia/reperfusion by left middle cerebral artery occlusion for 45 min using an intraluminal thread. Two days later male mice randomly received vehicle, MHP36 cells, MHP36-Dax1KD cells, and MHP36 cells suspended in 17β-estradiol (100 nm) or 17β-estradiol alone (100 nm) with serial behavioral testing over 28 days followed by post-mortem histology and blinded analysis. Recovery of sensorimotor function was accelerated and enhanced, and lesion volume was reduced by MHP36-Dax1KD transplants. Regarding mechanisms, immunofluorescence indicated increased synaptic plasticity and neuronal differentiation after MHP36-Dax1KD transplants. In conclusion, knockdown of Dax-1 is a useful target to increase 17β-estradiol biosynthesis in NSCs and improves functional recovery after stroke in vivo, possibly mediated through neuroprotection and improved synaptic plasticity. Therefore, targeting 17β-estradiol biosynthesis in stem cells may be a promising therapeutic strategy for enhancing the efficacy of stem cell-based therapies for stroke.

In ovo injection of betaine promotes adrenal steroidogenesis in pre-hatched chicken fetuses

Corticosterone is critical for the maturation and survival of chicken fetus around hatching. Betaine is used as a feed additive in poultry industry to promote growth and mitigate stress. However, it remains unknown whether betaine could affect adrenal corticosterone synthesis in pre-hatching chicken fetuses. In this study, betaine (2.5 mg/egg) was injected into developing chicken fetuses at d 11 of incubation (E11) and its impact on adrenal steroidogenesis was investigated at day 19 (E19). Plasma corticosterone concentration was significantly (P < 0.05) elevated in betaine-treated fetuses, together with increased adrenal expression of melanocortin 2 receptor and steroidogenic acute regulatory protein. Accordingly, the corticosterone biosynthetic enzymes, such as cytochrome P450 family 11 subfamily A member 1, 3β-hydroxysteroid dehydrogenase and cytochrome P450 family 21 subfamily A member 2, as well as cholesterol biosynthesis or regulation-related genes, such as sterol regulatory element-binding protein 1, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and low-density lipoprotein receptor, were all significantly (P < 0.05) upregulated in betaine group. Meanwhile, steroidogenic factor-1 and glucocorticoid receptor were significantly (P < 0.05) enhanced, whereas expression of dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene, a nuclear receptor known as a repressor of adrenal steroidogenesis, was significantly (P < 0.05) downregulated. Betaine significantly (P < 0.05) increased adrenal expression of genes involved in one-carbon metabolism and DNA methylation, such as S-adenosyl homocysteine hydrolase, betaine-homocysteine-methyltransferase, methionine adenosyl transferase and DNA methyltransferases, yet the promoter regions of most steroidogenic genes were significantly (P < 0.05) hypomethylated. These results indicate that in ovo injection of betaine promotes adrenal glucocorticoid synthesis in chicken fetuses before hatching, which involves alterations in DNA methylation.

TNF-α-mediated suppression of Leydig cell steroidogenesis involves DAX-1.

The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) has an inhibitory role in gonadal functions particularly in the steroidogenesis of Leydig cells. A detailed understanding of the mechanisms by which TNF-α regulates testicular steroidogenesis will be helpful in the design of novel clinical interventions for the treatment and prevention of male reproductive disorders. Here, we report that TNF-α-mediated activation of DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is involved in the inhibition of Leydig cell steroidogenesis. Rat testis Leydig tumor cells (LC-540) were treated with TNF-α (10 ng/ml) for different time intervals. To elucidate the pathways of intracellular signal transduction that regulate DAX-1 expression, we utilized specific inhibitors. The siRNA transfection of DAX-1 into LC-540 cells was performed by electroporation. The mRNA and protein levels were determined by RT-PCR and Western blotting, respectively. We found that the mRNA and protein levels of DAX-1 were increased by threefold approximately in TNF-α-treated cells when compared to controls. Staurosporine, JNK inhibitor SP600125 and ERK inhibitor PD98059 significantly decreased DAX-1 expression in TNF-α-treated Leydig cells when compared to their respective controls. Further, a siRNA-mediated knockdown of DAX-1 restores the expression of steroidogenic proteins in TNF-α-treated Leydig cells. These findings provide valuable information that TNF-α activates DAX-1 through JNK/ERK MAP kinase pathway which regulates the expression of steroidogenic enzyme genes in Leydig cells.

DAX-1 Inhibits Hepatocellular Carcinoma Proliferation by Inhibiting β-Catenin Transcriptional Activity

Background/Aims: Hepatocellular carcinoma (HCC) represents the most common type of liver cancer. DAX1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1), an atypical member of the nuclear receptor family due to lack of classical DNA-binding domains, has been known for its fundamental roles in the development, especially in the sex determination and steroidogenesis. Previous studies also showed that DAX-1 played a critical role in endocrine and sex steroid-dependent neoplasms such as adrenocortical, pituitary, endometrial, and ovarian tumors. However, its biological roles in the development of HCC remain largely unexplored. Methods: Real-time PCR and Western blot were used to detect the expression of DAX-1 in HCC tissues and cell lines. Immunoprecipitation (IP) assay was used to show the interaction between DAX-1 and β-Catenin. Small interfering RNA (siRNA) was used to silence the expression of DAX-1. BrdU incorporation and Cell-cycle assays were used to detect the role of DAX-1 in HCC cells proliferation. Migration and invasion assays were carried out to test the metastasis ability of DAX-1 in HCC cells. Results: In the present study, we found that mRNA and protein levels of DAX-1 were down-regulated in HCC tissues and cell lines. Furthermore, overexpression of DAX-1 could inhibit while its knockdown using small interfering RNA promoted cell proliferation in several HCC cell lines. At the molecular level, we demonstrated that DAX-1 could interact with β-Catenin and attenuate its transcriptional activity. Conclusion: Therefore, our results suggest a previously unknown DAX-1/β-Catenin molecular network controlling HCC development.

MicroRNA-561 Promotes Acetaminophen-Induced Hepatotoxicity in HepG2 Cells and Primary Human Hepatocytes through Downregulation of the Nuclear Receptor Corepressor Dosage-Sensitive Sex-Reversal Adrenal Hypoplasia Congenital Critical Region on the X Chromosome, Gene 1 (DAX-1)

One of the major mechanisms involved in acetaminophen (APAP)-induced hepatotoxicity is hepatocyte nuclear factor 4α (HNF4α)-mediated activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). In the present study, we investigated the role of miR-561 and its target gene DAX-1 encoding a corepressor of HNF4α in the process of APAP-induced hepatotoxicity. We used both human hepatocellular liver carcinoma cell line (HepG2) cells and primary human hepatocytes in this study and monitored the levels of reactive oxygen species, lactate dehydrogenase, and glutathione. Our bioinformatics study suggests an association between miR-561 and DAX-1, but not HNF4α. Treatment of HepG2 cells with APAP significantly reduced the expression of DAX-1 in a concentration-dependent manner. miR-561 was induced by APAP treatment in HepG2 cells. Transfection of HepG2 cells with an miR-561 mimic exacerbated APAP-induced hepatotoxicity. HNF4α is physically associated with DAX-1 in HepG2 cells. A decreased protein level of DAX-1 by APAP treatment was also enhanced by miR-561 mimic transfection in HepG2 cells and primary human hepatocytes. The basal and APAP-induced expression of PXR and CAR was enhanced by miR-561 mimic transfection; however, transfection of HepG2 cells or primary human hepatocytes with a miR-561 inhibitor or DAX-1 small interfering RNA reversed these effects. Additionally, the chromatin immunoprecipitation assay revealed that recruitment of DAX-1 onto the PXR promoter was inversely correlated with the recruitment of peroxisome proliferator-activated receptor-α coactivator-1α and HNF4α on APAP treatment. These results indicate that miR-561 worsens APAP-induced hepatotoxicity via inhibition of DAX-1 and consequent transactivation of nuclear receptors.

X-linked congenital adrenal hypoplasia associated with hypospadias in an Egyptian baby: a case report

IntroductionX-linked congenital adrenal hypoplasia is a rare developmental disorder of the human adrenal cortex and is caused by deletion or mutation of the dosage-sensitive sex reversal adrenal hypoplasia congenita critical region of the X chromosome, gene 1 (DAX-1) gene. Most affected children present with failure to thrive, salt wasting and hypoglycemic convulsions in the first months of life. Hypospadias affects approximately one in 250 live male births. Mutations in the mastermind-like domain-containing 1 (MAMLD1) gene have been implicated as one of the causes of hypospadias in children. To the best of our knowledge, an association between congenital adrenal hypoplasia due to a DAX-1 mutation and hypospadias due to mutation of the MAMLD1 gene has not previously been reported in the literature.Case presentationA 35-day-old male Egyptian baby was referred to our institution for the evaluation of a two-week history of recurrent vomiting associated with electrolyte imbalance. On examination, our patient was found to have hypotension and dehydration. A genital examination showed distal penile hypospadias with chordee and normal testes. He had hyponatremia, hyperkalemia, hypoglycemia and metabolic acidosis. Endocrinological investigations revealed low levels of cortisol, 17-hydroxyprogesterone and aldosterone, with a high level of adrenocorticotrophic hormone. A provisional diagnosis of congenital adrenal hypoplasia associated with hypospadias was made. A molecular genetics study confirmed the diagnosis of X-linked congenital adrenal hypoplasia due to DAX-1 mutations and hypospadias due to MAMLD1 mutation. He was started on hydrocortisone and fludrocortisone treatment. After three weeks of treatment, his symptoms improved and his blood sugar, sodium, potassium and cortisol levels normalized.ConclusionsWe report the case of an Egyptian baby with an association of congenital adrenal hypoplasia due to DAX-1 mutation and hypospadias due to MAMLD1 mutation. Early diagnosis of this association and determining its optimal treatment are vital in helping to avoid its fatal course.

The DAX1 mutation in a patient with hypogonadotropic hypogonadism and adrenal hypoplasia congenita causes functional disruption of induction of spermatogenesis

Hypogonadotropic hypogonadism (HH) associated with adrenal hypoplasia congenita (AHC) is a very rare syndrome caused by mutation of DAX1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome) [19]. The DAX1 is located on the chromosome X (Xp21.3–21.2) and contains two exons. It encodes a 470-amino acid protein which belongs to the nuclear hormone receptor superfamily (called DAX1). DAX1 is expressed in the adrenal cortex, pituitary and hypothalamus, gonadal cells such as Leydig and Sertoli cells, theca and granulosa cells and in germ cells [15, 26]. Patients with HH due to DAX1 mutation exhibited azoospermia [4, 5, 17, 24]. Results of spermatogenesis induction using exogenous gonadotropin are unsatisfactory [4, 17, 24]. However, Frapsauce et al. [5] has recently presented the first birth after successful assisted reproductive technique (ART) using TESE-ICSI in a man with HH and AHC linked to a DAX1 mutation. In this case, long period of gonadotropin treatment allowed for development of few mature spermatozoa obtained from testicular biopsy and used for ICSI [5]. Here, we report clinical and immunohistochemical studies of patient with HH and AHC due to deletion of the second exon of the DAX1 in order to induce spermatogenesis by gonadotropins treatment.

Characteristic induction of steroidogenic factor 1 (SF-1) and DAX-1 and enhanced expression of glucocorticoid synthesis-related genes in adrenals from spontaneously hypertensive rats

To gain insight into the molecular mechanism of hyper-glucocorticoidism in spontaneously hypertensive rats (SHR), this study investigated the expression of genes related to glucocorticoid synthesis, melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage enzyme (P450scc) and 11β-hydroxylase (P450c11), and the transcription factors of steroidogenic factor 1 (SF-1), which stimulates expression of the above gene, and DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1), which negatively regulates the transcriptional activity of SF-1, in adrenals from SHR. On quantitative real time RT-PCR analysis, gene expression levels of MC2R, StAR, P450scc and P450c11 in SHR were high compared with those in normotensive Wistar Kyoto rats (WKY). The gene expression level of SF-1 was not different between the two rats. However, the expression level of DAX-1 in SHR was markedly lower than that in WKY. Furthermore, the protein levels of StAR, SF-1 and DAX-1 determined by Western blot analysis coincided well with the gene expressions in both rats. These results suggest that the low level of DAX-1 may enhance the transcriptional activity of SF-1 and expression of genes related to glucocorticoid synthesis, which are targeted by SF-1, in adrenals from SHR.

Aldosterone-Producing Adenoma Formation in the Adrenal Cortex Involves Expression of Stem/Progenitor Cell Markers

Aldosterone producing adenoma (APA) is the most common form of surgically curable hypertension. To further understand mechanisms involved in APA formation, we investigated the expression of molecules linked to adrenal stem/precursor cells [β-catenin, Sonic hedgehog (Shh), CD56], and nuclear receptors that play key roles in adrenocortical development and function steroidogenic factor 1, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1) in six control adrenal glands and 14 adrenals with APA and compared their expression with that of specific markers of zona glomerulosa (ZG) [CYP11B2, Disabled 2 (Dab2)]. Both Dab2 and CD56 were expressed in ZG. Although Dab2 associates uniquely with differentiated ZG cells and its expression is lost when cells transdifferentiate to zona fasciculata (ZF) cells, CD56 was also expressed in ZF and in aldosterone-producing cell clusters, confirming that these structures possess an intermediate phenotype between ZG and ZF cells. Shh was barely detectable in cells located to the outer part of the ZG in the control adrenal; in contrast, its expression was detected in the entire APA and was dramatically increased in the hyperplastic peritumoral ZG. Transcriptome profiling revealed differential expression of components of Shh signaling pathway in a subgroup of APA. Similarly, Wnt/β-catenin signaling was activated in the majority of APA as well as in the entire peritumoral adrenal cortex; however, no mutation was identified in the CTNNB1 gene that could account for β-catenin activation. Our data suggest that both APA and adjacent ZG present characteristics of stem/precursor cells; the reexpression of genes involved in fetal adrenal development could underlie excessive ZG cell proliferation and APA formation.

DAX1 suppresses FXR transactivity as a novel co-repressor

Bile acid receptor FXR (farnesoid X receptor) is a key regulator of hepatic bile acid, glucose and lipid homeostasis through regulation of numerous genes involved in the process of bile acid, triglyceride and glucose metabolism. DAX1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is an atypical member of the nuclear receptor family due to lack of classical DNA-binding domains and acts primarily as a co-repressor of many nuclear receptors. Here, we demonstrated that DAX1 is co-localized with FXR in the nucleus and acted as a negative regulator of FXR through a physical interaction with FXR. Our study showed that over-expression of DAX1 down-regulated the expression of FXR target genes, whereas knockdown of DAX1 led to their up-regulation. Furthermore, three LXXLL motifs in the N-terminus of DAX1 were required for the full repression of FXR transactivation. In addition, our study characterized that DAX1 suppresses FXR transactivation via competing with co-activators such as SRC-1 and PGC-1α. In conclusion, DAX1 acts as a co-repressor to negatively modulate FXR transactivity.

DAX-1 and DAX-1A expression in human testicular tissues with primary spermatogenic failure

DAX-1 [dosage-sensitive sex reversal-adrenal hypoplasia congenital (AHC) critical region on the X chromosome gene 1; NR0B1] is an orphan nuclear receptor that acts as a transcriptional repressor in adrenal/gonadal development, steroidogenesis and probably spermatogenesis. An alternatively spliced form called DAX-1A (NR0B1A) has been described in several tissues including the testis, and in vitro studies have shown an inhibitory effect on DAX-1 transcriptional function. We aimed to study the mRNA and protein expression of DAX-1 in testicular tissues of 65 men with primary spermatogenic failure [complete Sertoli cell only syndrome (SCOS), focal SCOS, maturation arrest and mixed atrophy] compared with 33 controls with normal spermatogenesis. As a novel finding, we observed intense immunostaining, not only in the nucleus of Sertoli cells, but also in pachytene spermatocytes and round spermatids. The quantitative mRNA expression of DAX-1 and DAX-1A was similar between cases and controls and was not associated with the levels of gonadotrophins and steroids. Moreover, DAX-I transcript expression level was ∼750-fold higher than DAX-1A, and there was a strong positive correlation between them (r = 0.52; P< 0.001). We conclude that, in addition to Sertoli cells, DAX-1/DAX-1A is expressed in germ cells from spermatogonia to round spermatids. Besides, the similar mRNA expression of DAX-I and DAX-IA in testicular tissues from cases and controls does not support the involvement of DAX-1 in the etiology of primary spermatogenic failure. Finally, the low level of expression of the alternative transcriptional variant DAX-1A would not support its putative inhibitory function in vivo.

Effects of apigenin on steroidogenesis and steroidogenic acute regulatory gene expression in mouse Leydig cells

Previous studies reported that the age-related decline in testosterone biosynthesis is associated with a decrease in the steroidogenic acute regulatory (StAR) protein which regulates the rate-limiting step of testosterone biosynthesis. To explore the possibility of delaying this decline using a dietary approach, we have examined the effect of a natural flavonoid, apigenin, on StAR gene expression in mouse Leydig cells. Incubation of these cells with the flavonoid enhanced cyclic adenosine monophosphate (cAMP)-induced steroidogenesis and StAR protein expression. The results from the analyses of StAR mRNA by reverse transcription-polymerase chain reaction and the luciferase assays of StAR promoter activity indicated that this flavonoid enhanced StAR gene expression at the level of transcription. Further studies showed that apigenin blocked the thromboxane A2 receptor and interrupted the signaling through the cyclooxygenase-2-thromboxane A synthase-thromboxane A2-receptor pathway, resulting in a reduction of DAX-1 (dosage sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene-1) protein, a transcriptional repressor of StAR gene expression. When DAX-1 protein was reduced, the sensitivity of the Leydig cells was dramatically enhanced, with sub-threshold level of cAMP being able to induce maximal levels of StAR protein expression and steroid hormone production. The present study suggests a potential application of apigenin to improve StAR protein expression and steroidogenic sensitivity of aging Leydig cells.

Orphan Nuclear Receptor DAX-1 Acts as a Novel Corepressor of Liver X Receptor α and Inhibits Hepatic Lipogenesis

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is a member of the nuclear receptor superfamily that can repress diverse nuclear receptors and has a key role in adreno-gonadal development. Our previous report has demonstrated that DAX-1 can inhibit hepatocyte nuclear factor 4alpha transactivity and negatively regulate gluconeogenic gene expression (Nedumaran, B., Hong, S., Xie, Y. B., Kim, Y. H., Seo, W. Y., Lee, M. W., Lee, C. H., Koo, S. H., and Choi, H. S. (2009) J. Biol. Chem. 284, 27511-27523). Here, we further expand the role of DAX-1 in hepatic energy metabolism. Transfection assays have demonstrated that DAX-1 can inhibit the transcriptional activity of nuclear receptor liver X receptor alpha (LXRalpha). Physical interaction between DAX-1 and LXRalpha was confirmed Immunofluorescent staining in mouse liver shows that LXRalpha and DAX-1 are colocalized in the nucleus. Domain mapping analysis shows that the entire region of DAX-1 is involved in the interaction with the ligand binding domain region of LXRalpha. Competition analyses demonstrate that DAX-1 competes with the coactivator SRC-1 for repressing LXRalpha transactivity. Chromatin immunoprecipitation assay showed that endogenous DAX-1 recruitment on the SREBP-1c gene promoter was decreased in the presence of LXRalpha agonist. Overexpression of DAX-1 inhibits T7-induced LXRalpha target gene expression, whereas knockdown of endogenous DAX-1 significantly increases T7-induced LXRalpha target gene expression in HepG2 cells. Finally, overexpression of DAX-1 in mouse liver decreases T7-induced LXRalpha target gene expression, liver triglyceride level, and lipid accumulation. Overall, this study suggests that DAX-1, a novel corepressor of LXRalpha, functions as a negative regulator of lipogenic enzyme gene expression in liver.

Characterisation of Estrogen Responses in Breast Cancer Cells Highlights LRH-1 as a New Target for Breast Cancer Treatment.

Abstract Background: Liver receptor homolog-1 (LRH-1; NR5A2) is an orphan member of the Ftz-F1 family of nuclear receptors, which comprises four members (NR5A1-NR5A4). It is expressed in endodermal derived tissues and has been linked to a number of developmental, metabolic and proliferative processes. LRH-1 plays an important role in cholesterol and lipid homeostasis. Recent findings have shown that intestinal production of glucocorticoids is regulated by LRH-1, a deficiency of which sensitizes mice to colitis. Uncontrolled LRH-1 activity participates in the induction of intestinal tumours. LRH-1 protein expression has also been detected by immunohistochemistry in tumour cells of human mammary ductal carcinomas.Materials and Methods: Using a panel of breast cancer cell lines and human tissues we have studied the expression of LRH-1 together with another Ftz-F1 subfamily member steroidogenic factor 1 (SF-1) and the potent LRH-1 co-repressors small heterodimer partner (SHP) and DAX-1/Dax-1 (dosage-sensitive sex-reversal adrenal hypoplasia congenital critical region on the X chromosome 1). RNA ligase Mediated Rapid amplification of 5' complementary DNA ends (5'RLM-RACE) was performed in order to map the estrogen regulated transcription start sites for the LRH-1 gene. No natural ligand for LRH-1 has been described. Using fluorescence resonance energy transfer (FRET)-based assays a series of unusual bicyclic compounds have been defined as potent LRH-1 ligands. The effects of these compounds on breast cancer cell growth and gene expression has been investigated using SRB growth assays and quantitative RT-PCR.Results &amp; Discussion: LRH-1 expression was found to be highest in estrogen receptor positive cell lines and breast, colon,small intestine and liver tissue. Treatment with 17β-estradiol increased LRH-1 levels in estrogen receptor positive cell lines, while treatment with anti-estrogens Tamoxifen and ICI 182,780 (Fulvestrant) reduced LRH-1 levels. siRNA mediated RNA interference against LRH-1 significantly reduced the estrogen-dependent growth of estrogen receptor positive breast cancer cell lines. We have shown that LRH-1 regulates the expression of the LRH-1 target gene Inhibin-α (a member of the transforming growth factor-β superfamily) in breast cancer cell lines. 5' RACE identified two new variant forms of LRH-1, which we have named LRH-1 289 and LRH-1 325 respectively. These arise from a novel promoter region lying between exon 2 and exon 3. RT- PCR for the LRH-1 289 variant showed E2 regulation in MCF7 cells and was seen to account for the majority of LRH-1 expression in this line. SRB growth assays demonstrated that these bicyclical compounds stimulate the growth of estrogen receptor/LRH-1 positive breast cancer cell lines in a dose dependent manner. Luciferase assays have demonstrated a titratable increase in LRH-1 reporter activity following the addition of these compounds.Our studies show that LRH-1 is a potently estrogen regulated gene in breast cancer cells, which acts to regulate cell growth and gene expression. These results therefore help validate LRH-1 as a potential new target for breast cancer treatment Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4143.