Adrenal Hypoplasia Congenita Critical Region Research Articles

Identification and involvement of DAX1 gene in spermatogenesis of boring giant clam Tridacna crocea

DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenital critical region on X chromosome gene 1), a key sex determinant in various species, plays a vital role in gonad differentiation and development and controls spermatogenesis. However, the identity and function of DAX1 are still unclear in bivalves. In the present study, we identified a DAX1 (designed as Tc-DAX1) gene from the boring giant clam Tridacna crocea, a tropical marine bivalve. The full length of Tc-DAX1 was 1877 bp, encoding 462 amino acids, with a Molecular weight of 51.81 kDa and a theoretical Isoelectric point of 5.87 (pI). Multiple sequence alignments and phylogenetic analysis indicated a putative ligand binding domain (LBD) conserved regions clustered with molluscans DAX1 homologs. The tissue distributions in different reproductive stages revealed a dimorphic pattern, with the highest expression trend in the male reproductive stage, indicating its role in spermatogenesis. The DAX1 expression data from embryonic stages shows its highest expression profile (P < 0.05) in the zygote stage, followed by decreasing trends in the larvae stages (P > 0.05). The localization of DAX1 transcripts has also been confirmed by whole mount in situ hybridization, showing high positive signals in the fertilized egg, 2, and 4-cell stage, and gastrula. Moreover, RNAi knockdown of the Tc-DAX1 transcripts shows a significantly lower expression profile in the ds-DAX1 group compared to the ds-EGFP group. Subsequent histological analysis of gonads revealed that spermatogenesis was affected in a ds-DAX1 group compared to the ds-EGFP group. All these results indicate that Tc-DAX1 is involved in the spermatogenesis and early embryonic development of T. crocea, providing valuable information for the breeding and aquaculture of giant clams.

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.

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.

Induced Pluripotent Stem Cells from a Marsupial, the Tasmanian Devil (Sarcophilus harrisii): Insight into the Evolution of Mammalian Pluripotency.

We demonstrate the generation of Tasmanian devil (Sarcophilus harrisii) induced pluripotent stem cells (DeviPSCs) from dermal fibroblasts by lentiviral delivery of human transcription factors. DeviPSCs display characteristic pluripotent stem cell colony morphology, with individual cells having a high nuclear-to-cytoplasmic ratio and alkaline phosphatase activity. DeviPSCs are leukemia inhibitory factor dependent and have reactivated endogenous octamer-binding transcription factor 4 [OCT4, POU domain, class 5, transcription factor 1 (POU5F1)], POU2 [POU domain, class 5, transcription factor 3 (POU5F3)], sex determining region Y-box 2 (SOX2), Nanog homeobox (NANOG) and dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1) genes, retained a normal karyotype, and concurrently silenced exogenous human transgenes. Notably, co-expression of both OCT4 and POU2 suggests that they are representative of cells of the epiblast, the marsupial equivalent of the inner cell mass. DeviPSCs readily form embryoid bodies and in vitro teratomas containing derivatives of all three embryonic germ layers. To date, DeviPSCs have been stably maintained for more than 45 passages. Our DeviPSCs provide an invaluable resource for studies into marsupial pluripotency and development, and they may also serve as an important tool in efforts to combat the threat of devil facial tumor disease.

Gender-specific differences in gene expression profiles in gynogenetic Pengze crucian carp

Gynogenesis is a form of asexual reproduction that is used to obtain all-female fish stocks. In this study, we were interested in studying gender-specific differences in gene expression profiles in gynogenetic teleosts, using a carp species. The four-month old gynogenetic Pengze crucian carp F1 (Carassius auratus var. pengzensis, Pcc) showed a high ratio of males under laboratory culture condition. The present study aimed to investigate the differences between males and females. The gonadosomatic index of the females was significantly higher than that of the males. Moreover, the hepatosomatic index of the females was significantly lower than that of the males. Vitellogenin B mRNA was abnormally highly expressed in male hepatopancreas and testes compared to females. Similarly, zona pellucida 2 expressed at a significantly high level in the testes. For the sex related genes, dosage-sensitive sex reversal, adrenal hypoplasia congenital critical region on the X-chromosome gene 1, doublesex and mab-3 related transcription factor 1a, nuclear receptor subfamily 5, group A, member 1b and SRY-box containing gene 9a had significantly higher expression levels in the males than in the females, whereas there was no difference in expression of anti-Müllerian hormone, cytochrome P450 family 19 subfamily A member 1A and forkhead box L2 transcripts between the two genders. The females showed higher levels of estrogen but no significant difference in testosterone compared to the males. The data suggest remarkable differences between the two genders of the Pengze crucian carp.

Primary Adrenal Insufficiency in a Newborn With Adrenal Hypoplasia Congenita Caused by a Mutation of theDAX1Gene

Adrenal hypoplasia congenita (AHC) is a rare inherited disorder of the adrenal gland caused by deletion or mutation of the dosage-sensitive sex-reversal AHC critical region on the X chromosome, gene 1 (DAX1) gene. The DAX1 gene is expressed in the adrenal cortex, the pituitary gland, the hypothalamus, the testis, and the ovary. Most affected infants present with failure to thrive, salt wasting, and hypoglycemic seizure in early life. Immediate mineralocorticoid and glucocorticoid replacement is essential. Most boys with AHC present with hypogonadotropic hypogonadism, resulting in failure to enter puberty and the need for testosterone treatment. However, a recent study revealed that the onset of puberty in boys with AHC can be variable, ranging from arrested or absent to precocious. We describe a case involving a newborn who presented with primary adrenal insufficiency due to a mutation of the DAX1 gene and was finally diagnosed with AHC.

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.

Minireview: Role Of Orphan Nuclear Receptors in Cancer and Potential as Drug Targets

The nuclear orphan receptors for which endogenous ligands have not been identified include nuclear receptor (NR)0B1 (adrenal hypoplasia congenita critical region on chromosome X gene), NR0B2 (small heterodimer partner), NR1D1/2 (Rev-Erbα/β), NR2C1 (testicular receptor 2), NR2C2 (testicular receptor 4), NR2E1 (tailless), NR2E3 (photoreceptor-specific NR [PNR]), NR2F1 chicken ovalbumin upstream promoter transcription factor 1 (COUP-TFI), NR2F2 (COUP-TFII), NR2F6 (v-erbA-related protein), NR4A1 (Nur77), NR4A2 (Nurr1), NR4A3 (Nor1), and NR6A1 (GCNF). These receptors play essential roles in development, cellular homeostasis, and disease including cancer where over- or underexpression of some receptors has prognostic significance for patient survival. Results of receptor knockdown or overexpression in vivo and in cancer cell lines demonstrate that orphan receptors exhibit tumor-specific pro-oncogenic or tumor suppressor-like activity. For example, COUP-TFII expression is both a positive (ovarian) and negative (prostate and breast) prognostic factor for cancer patients; in contrast, the prognostic activity of adrenal hypoplasia congenita critical region on chromosome X gene for the same tumors is the inverse of COUP-TFII. Functional studies show that Nur77 is tumor suppressor like in acute leukemia, whereas silencing Nur77 in pancreatic, colon, lung, lymphoma, melanoma, cervical, ovarian, gastric, and some breast cancer cell lines induces one or more of several responses including growth inhibition and decreased survival, migration, and invasion. Although endogenous ligands for the orphan receptors have not been identified, there is increasing evidence that different structural classes of compounds activate, inactivate, and directly bind several orphan receptors. Thus, the screening and development of selective orphan receptor modulators will have important clinical applications as novel mechanism-based agents for treating cancer patients overexpressing one or more orphan receptors and also for combined drug therapies.

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.

Detection of Carrier Status and Mutations in Family Members of a Child with Complete Deletion of the DAX1 Gene using Multiplex Ligation-Dependent Probe Amplification

To the Editor:We have previously reported a case of adrenal crisis [1]. Real-time SYBR Green DNA PCR and gel electrophoresis showed lack of amplification of exons of NRB01/DAX1 (dosage-sensitive sex reversal) gene on Xp21 locus, suggesting a complete deletion. We demonstrated that molecular techniques may prove useful in making a diagnosis and for genetic counselling in adrenal hypoplasia congenital (AHC); information in this letter is supplementary to the article already published [1]. AHC is a rare congenital adrenal disorder where there is primary adrenal failure in early life and hypogonadotropic hypogonadism during adolescence. X-linked AHC is caused by deletions/mutations in DAX1 gene AHC critical region on X chromosome. We performed genetic analysis using Multiplex Ligation-Dependent Probe Amplification (MLPA) on family members of proband, so as to further understand genetic factors involved in inheritance and presentation of this child with AHC, and to detect affected or carrier status in relatives. MLPA, an extension of PCR, allows highly multiplexed detection of relative copy number variations of target sequences in a fast, reliable, and cost-effective manner [2]. Genetic studies in families of patients with affected members have been previously reported [3]. Six family members were tested by MLPA: child’s parents, sister, maternal aunt and her children (Fig. 1). MLPA analysis was performed using SALSA MLPA P185 Intersex probemix (MRC-Holland, Netherlands). Each probe generates an amplification product of unique length that can be detected by capillary electrophoresis. Comparison of relative sizes of the fluorescent peaks from the target probes with reference probes coamplified during the reaction, using analysis software, allows quantification of relative abundance of target regions. N. D. Phadke :K. A. Khatod GenePath Dx (Causeway Healthcare Pvt Ltd), J.M. Road, Shivajinagar, Pune, India

Duplication of dosage sensitive sex reversal area in a 46, XY patient with normal sex determining region of Y causing complete sex reversal

The sex chromosome composition of the primordial gonad, either 46XX or 46XY, determines its differentiation as ovaries or testes. Local hormones secreted by developing gonads and tissue specific transcription factors influence the differentiation of external and internal genital structures. Dosage sensitive sex reversal adrenal hypoplasia congenita critical region (DAX1) on Xp21 is a gene which is expressed in the developing adrenals, gonads, hypothalamus and pituitary gland. Duplication of this area causes dosage sensitive male-to-female sex reversal while mutation or deletion leads to adrenal hypoplasia congenita with hypogonadotropic hypogonadism in affected males. To report a case with duplication of the X chromosome segment within the region of Xp21.1-22.2 resulting in 46 XY sex reversal and a literature review on DAX1 and dosage sensitive sex reversal (DSS). We present the clinical history, physical findings, laboratory, and imaging study results in a newborn baby. This infant was sex assigned as female at birth and had normal female external genitalia. Chromosome analysis was done due to multiple minor malformations and showed a karyotype of 46 Xp+Y. Fluorescent in situ hybridization analysis revealed the duplication in the DSS area. Duplication of the DAX1 gene on the X chromosome with normal sex determining region of Y (SRY) results in 46 XY sex reversal. This was inherited from the mother who had normal ovarian function. Additional problems include growth failure, mental retardation and multiple congenital anomalies. The baby did not have a mutation or deletion of DAX1, which would have caused adrenal insufficiency and hypogonadism.

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.

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.

Seven NovelDAX1Mutations with Loss of Function Identified in Chinese Patients with Congenital Adrenal Hypoplasia

DAX1 (for dosage-sensitive sex reversal, adrenal hypoplasia congenital critical region on the X chromosome, gene 1; also called NROB1) mutations are responsible for adrenal failure and hypogonadotropic hypogonadism in patients with adrenal hypoplasia congenita (AHC), through a loss of trans-repression of SF-1 (for steroidogenic factor-1)-mediated StAR (for steroidogenic acute regulatory protein) and LHbeta transcriptional activities and a reduction of GnRH expression. The correlation of clinical features with genetic and functional alterations of the gene was investigated in detail in AHC patients. The present study aimed at identifying DAX1 mutations in Chinese AHC patients and investigating the functional defects of detected novel mutations. Nine patients with AHC were recruited from eight families. DAX1 mutations were screened, and the transcriptional activities of the identified mutations were assessed in vitro. DAX1 mutations were detected in all nine patients enrolled in the study, with eight different mutations. Among the latter, seven are novel mutations, including two missense (L262P and C368F), one nonsense (Q222X), and four frame-shift (637delC, 652_653delAC, 973delC, and 774_775insCC) mutations. The functional studies showed that the mutant DAX1 was impaired by nuclear localization, loss of trans-repression of StAR and LHbeta transcriptional activities, and reduction of GnRH expression. These findings provide insight into the molecular events by which DAX1 mutations influence the hypothalamus-pituitary-gonadal and hypothalamus-pituitary-adrenal axis and lead to AHC and hypogonadotropic hypogonadism.

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.

DAX-1 Acts as a Novel Corepressor of Orphan Nuclear Receptor HNF4α and Negatively Regulates Gluconeogenic Enzyme Gene Expression

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is an atypical member of the nuclear receptor family and acts as a corepressor of a number of nuclear receptors. HNF4alpha (hepatocyte nuclear factor 4alpha) is a liver-enriched transcription factor that controls the expression of a variety of genes involved in cholesterol, fatty acid, and glucose metabolism. Here we show that DAX-1 inhibits transcriptional activity of HNF4alpha and modulates hepatic gluconeogenic gene expression. Hepatic DAX-1 expression is increased by insulin and SIK1 (salt-inducible kinase 1), whereas it is decreased in high fat diet-fed and diabetic mice. Coimmunoprecipitation assay from mouse liver samples depicts that endogenous DAX-1 interacts with HNF4alpha in vivo. In vivo chromatin immunoprecipitation assay affirms that the recruitment of DAX-1 on the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is inversely correlated with the recruitment of PGC-1alpha and HNF4alpha under fasting and refeeding, showing that DAX-1 could compete with the coactivator PGC-1alpha for binding to HNF4alpha. Adenovirus-mediated expression of DAX-1 decreased both HNF4alpha- and forskolin-mediated gluconeogenic gene expressions. In addition, knockdown of DAX-1 partially reverses the insulin-mediated inhibition of gluconeogenic gene expression in primary hepatocytes. Finally, DAX-1 inhibits PEPCK and glucose-6-phosphatase gene expression and significantly lowers fasting blood glucose level in high fat diet-fed mice, suggesting that DAX-1 can modulate hepatic gluconeogenesis in vivo. Overall, this study demonstrates that DAX-1 acts as a corepressor of HNF4alpha to negatively regulate hepatic gluconeogenic gene expression in liver.

Gene expression patterns in juvenile American alligators ( Alligator mississippiensis) exposed to environmental contaminants

Reproductive and developmental abnormalities have been reported in the American alligator ( Alligator mississippiensis) population from Lake Apopka, FL, that is chronically exposed to a complex mixture of environmental contaminants. To begin to understand the molecular mechanisms that could lead to the observed abnormalities of the reproductive and endocrine system, we quantified concentrations of the steroid hormones testosterone (T) and estradiol-17β (E 2) and expression of steroid hormone receptors and genes relating to steroidogenesis in gonadal tissue from juvenile alligators from three lakes in Florida using enzyme immunoassay and quantitative real-time polymerase chain reaction. Alterations of ESR2 (estrogen receptor β) and SF1 (steroidogenic factor 1) mRNA expression in male gonadal tissue, without an observed difference in plasma concentrations of T, from the different lakes, begin to provide insight into potential mechanisms underlying the alterations of the reproductive system previously observed. Likewise, alterations in P450 aromatase and DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1) mRNA expression, with elevated plasma E 2 concentrations in females, provide leads to the potential mechanisms modifying folliculogenesis and ovarian development. The investigation of these genes also helps clarify normal endocrine and reproductive system function in the American alligator.

Four Japanese Patients with Adrenal Hypoplasia Congenita and Hypogonadotropic Hypogonadism Caused by DAX-1 Gene Mutations: Mutant DAX-1 Failed to Repress Steroidogenic Acute Regulatory Protein (StAR) and Luteinizing Hormone .BETA.-subunit Gene Promoter Activity

Mutations of DSS (dosage sensitive sex reversal)-AHC critical region on the X chromosome, gene 1 DAX-1(NROB1)] results in X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HHG). Here we report four Japanese patients with AHC and HHG caused by the mutations of the DAX-1 gene. All patients manifested adrenal crisis at early childhood. Three patients did not show any pubertal sign and were diagnosed as having HHG. One patient manifested spontaneous pubertal development at 17 years of age. Nevertheless, his puberty did not develop further and his gonadotropin and testosterone levels decreased thereafter. Therefore, he was also diagnosed as having HHG. We performed testicular biopsy in another patient with HHG. Histological examination demonstrated Sertoli cell hypoplasia and no sperm formation in the seminiferous tubules. Molecular analysis demonstrated two novel point mutations (V269D and L278R) in two patients. Transient transfection assays showed that all these mutations (V269D, L271X, L278R, and Q395X) abolished the repression activity to both StAR and LHbeta gene promoter activation. In conclusion, we reported patients with AHC and HHG caused by the loss of function mutations of the DAX-1 gene.