DAX-1 Expression Research Articles

The expression of nr0b1, the earliest gene in zebrafish tooth development, is a marker for human tooth and ameloblastoma formation

Zebrafish teeth develop on pharyngeal jaws in the 5th branchial arch, but early tooth development is remarkably similar to mammals (Borday-Birraux et al., Evol Dev 8:130, 2006). Recently, eve1 has been shown to be associated with the primary tooth (4V1) and early ameloblast development, the enamel organ precursor (Laurenti et al., Dev Dyn 230:727, 2004). dax1 is initially expressed in the 5th branchial arch in zebrafish at approximately 26 h postfertilization (hpf) and colocalizes with eve1 expression at ~48 hpf. Embryos injected with dax1 morpholino show downregulation of eve1 expression. Based on the zebrafish observations, we demonstrated novel DAX1 expression in normal human dental, benign ameloblastoma, and malignant ameloblastoma tissues. The association of NR0B1 and its protein product DAX1 with primary tooth development and ameloblastoma tumorigenesis is an association not previously described.

Dax-1 Knockdown in Mouse Embryonic Stem Cells Induces Loss of Pluripotency and Multilineage Differentiation

Dax-1 (Nr0b1) is an orphan member of the nuclear hormone receptor superfamily that has a key role in adrenogonadal development and function. Recent studies have also implicated Dax-1 in the transcriptional network controlling embryonic stem (ES) cell pluripotency. Here, we show that Dax-1 expression is affected by differentiating treatments and pharmacological activation of beta-catenin-dependent transcription in mouse ES cells. Furthermore, Dax-1 knockdown induced upregulation of multilineage differentiation markers, and produced enhanced differentiation and defects in ES viability and proliferation. Through RNA interference and transcriptome analysis, we have identified genes regulated by Dax-1 in mouse ES cells at 24 and 48 hours after knockdown. Strikingly, the great majority of these genes are upregulated, showing that the prevalent function of Dax-1 is to act as a transcriptional repressor in mouse ES cells, as confirmed by experiments using the Gal4 system. Genes involved in tissue differentiation and control of proliferation are significantly enriched among Dax-1-regulated transcripts. These data show that Dax-1 is an essential element in the molecular circuit involved in the maintenance of ES cell pluripotency and have implications for the understanding of stem cell function in both physiological (adrenal gland) and clinical (Ewing tumors) settings where Dax-1 plays a pivotal role in development and pathogenesis, respectively.

Histone H3 acetylation of StAR and decrease in DAX-1 is involved in the luteinization of bovine granulosa cells during in vitro culture

We investigated the expression of genes and transcription factors associated with steroidogenesis during the luteinization of granulosa cells isolated from bovine small follicles. Granulosa cells produced progesterone when cultivated in a culture medium including serum and attached to the substrate and began to display an elongated or fibroblastic aspect within 24 h of culture. We observed an increase in the number of granulosa cells at the same time. P450arom expression in the cultured granulosa cells had decreased at 24 h compared with 0 h of culture, and afterward was maintained at a low level. This expression was consistent with the decline of E2 concentration in the medium. Expression of StAR and P450scc mRNAs in the cultured granulosa cells was significantly increased at 72 h compared with 0 h of culture. Although the expression of Ad4BP/SF-1 mRNA began to increase during period between 48 and 72 h of culture, protein expression of Ad4BP/SF-1 remained at a constant level throughout the culture period. DAX-1 mRNA expression had decreased at 24 h of culture and remained at a low level. In parallel with this expression, the protein expression of DAX-1 began to decrease between 24 and 48 h of culture and then remained at a low level. Histone H3 acetylation of the StAR promoter region was observed at 72 h of culture period. Our data suggested that the decrease of Dax-1 transcription factor and the increase in histone H3 acetylation may play important roles in progesterone synthesis in luteinizing granulosa cells.

Inhibin A inhibits follicle-stimulating hormone (FSH) action by suppressing its receptor expression in cultured rat granulosa cells

Inhibin has long been considered as a suppresser of follicle-stimulating hormone (FSH) secretion from anterior pituitary through pituitary-gonad negative feedback to regulate follicle development. We demonstrated that addition of inhibin A could significantly suppress FSH-induced FSHR mRNA level in cultured rat granulosa cells (GCs) measured by real-time PCR. The inhibin A exerted its action mainly by inhibiting FSHR promoter activity. Furthermore, exogenous inhibin A could dramatically decrease FSH-induced P450arom and P450scc level and suppress progesterone and estradiol production in the cultured GCs, but it did not decrease forskolin-induced steroidogenesis, indicating that the inhibitory effect of inhibin A on FSH action may be upstream of cAMP signaling. Inhibin A was also capable of suppressing FSH-induced expression of steroidogenic factor 1 (SF-1) and androgen receptor, but stimulating DAX-1 expression in the culture. Our study has provided new evidence to show that inhibin A is capable of feedback antagonizing FSH action on GCs by reducing FSHR expression at ovarian level via a short feedback loop. Transcriptional factor receptors, such as SF-1, AR and DAX-1 were involved in this regulation.

Nuclear Receptor DAX1 in Human Prostate Cancer: A Novel Independent Biological Modulator

The orphan nuclear receptor DAX1 (dosage-sensitive sex reversal-AHC critical region on the X chromosome gene 1; NR0B1) has been known for its various roles in human development, specifically sex determination and steroidogenesis. Its expression has been reported in endocrine and sex steroid-dependent neoplasms such as human adrenocortical, pituitary, endometrial, and ovarian tumors. Prostate cancer is also sex steroid-dependent tumor in which androgens play important roles in the pathogenesis and development via androgen receptor (AR). DAX1 is also reported to repress AR activity in human prostate cancer cell line (LNCaP) but its biological roles have remained unclear in the human prostate cancer. The aim of this study is to examine the expression of DAX1 in human prostate cancer using immunohistochemistry in order to evaluate its possible biological and/or clinical significance. In this study, we examined the DAX1 immunoreactivity in human prostate cancer obtained from surgery (n = 40), and correlated the findings with clinicopathological features of the patients. Twenty-one cases were defined as positive cases for DAX1 immunoreactivity (53%). Immunoreactivity for DAX1 was inversely and significantly correlated with Gleason score (P<0.05). However, DAX1 immunoreactivity was not significantly correlated with the status of sex steroid receptors we examined. DAX1 immunoreactivity is considered a new biological modulator of human prostate cancer, but independent to the status of sex steroid receptors in human prostate cancer tissues.

DAX-1A (NR0B1A) Expression Levels are Extremely Low Compared to DAX-1 (NR0B1) in Human Steroidogenic Tissues

The orphan nuclear receptor DAX-1 (dosage-sensitive sex reversal-AHC critical region on the X chromosome gene 1; NR0B1) is known for its role in human development, specifically sex determination and steroidogenesis. Several recent publications have described an alternatively spliced form of DAX-1 called DAX-1A ( NR0B1A). DAX-1A is encoded by exons 1 and 2A of DAX-1, with exon 2A located within the DAX-1 intron 1. DAX-1A expression has been observed in several tissues, including adrenal gland, ovary, and testis. Transfection studies have further shown that DAX-1A has an inhibitory effect on DAX-1, suggesting a role for DAX-1A in the regulation of adrenal and gonadal differentiation/function. However, the relative level of DAX-1 versus DAX-1A transcripts still remains unclear. Herein, we developed and performed quantitative real-time RT-PCR to measure DAX-1 and DAX-1A mRNA expression levels in H295R human adrenal carcinoma cell lines, human adult and fetal adrenal glands, corpus luteum, testis, whole pre- and postmenopausal ovaries, ovarian follicles, placenta, liver, and kidney. These mRNA expression levels were quantified using DAX-1 and DAX-1A standard curves. In addition, Western blotting analysis was performed to examine both DAX-1 and DAX-1A protein levels in H295R cells, adrenal glands, corpus luteum, and liver. Both DAX-1 and DAX-1A mRNA were detected in all samples of H295R cells, human fetal and adult adrenals, testis, ovary, ovarian follicles, and corpus luteum. However, DAX-1 mRNA levels were significantly higher (> 37-fold) than that seen for DAX-1A (p<0.01). DAX-1A mRNA expression levels were undetectable in human liver, placenta, and kidney. Western blotting analysis results demonstrated that DAX-1 protein was predominantly expressed in H295R cells, human adult adrenal, and corpus luteum. These results suggest that in comparison to DAX-1A, DAX-1 is, by far, the predominant mRNA isoform found in human adrenal glands and gonads.

Role of Dosage-Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, Critical Region on the X Chromosome, Gene 1 in Protein Kinase A- and Protein Kinase C-Mediated Regulation of the Steroidogenic Acute Regulatory Protein Expression in Mouse Leydig Tumor Cells: Mechanism of Action

Dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) is an orphan nuclear receptor that has been demonstrated to be instrumental to the expression of the steroidogenic acute regulatory (StAR) protein that regulates steroid biosynthesis in steroidogenic cells. However, its mechanism of action remains obscure. The present investigation was aimed at exploring the molecular involvement of DAX-1 in protein kinase A (PKA)- and protein kinase C (PKC)-mediated regulation of StAR expression and its concomitant impact on steroid synthesis using MA-10 mouse Leydig tumor cells. We demonstrate that activation of the PKA and PKC pathways, by a cAMP analog dibutyryl (Bu)2cAMP [(Bu)2cAMP] and phorbol 12-myristate 13-acetate (PMA), respectively, markedly decreased DAX-1 expression, an event that was inversely correlated with StAR protein, StAR mRNA, and progesterone levels. Notably, the suppression of DAX-1 requires de novo transcription and translation, suggesting that the effect of DAX-1 in regulating StAR expression is dynamic. Chromatin immunoprecipitation studies revealed the association of DAX-1 with the proximal but not the distal region of the StAR promoter, and both (Bu)2cAMP and PMA decreased in vivo DAX-1-DNA interactions. EMSA and reporter gene analyses demonstrated the functional integrity of this interaction by showing that DAX-1 binds to a DNA hairpin at position -44/-20 bp of the mouse StAR promoter and that the binding of DAX-1 to this region decreases progesterone synthesis by impairing transcription of the StAR gene. In support of this, targeted silencing of endogenous DAX-1 elevated basal, (Bu)2cAMP-, and PMA-stimulated StAR expression and progesterone synthesis. Transrepression of the StAR gene by DAX-1 was tightly associated with expression of the nuclear receptors Nur77 and steroidogenic factor-1, demonstrating these factors negatively modulate the steroidogenic response. These findings provide insight into the molecular events by which DAX-1 influences the PKA and PKC signaling pathways involved in the regulation of the StAR protein and steroidogenesis in mouse Leydig tumor cells.

Evolution of the gene network underlying gonadogenesis in turtles with temperature-dependent and genotypic sex determination

The evolution of sex determination has long fascinated biologists, as it has paramount consequences for the evolution of a multitude of traits, from sex allocation to speciation and extinction. Explaining the diversity of sex-determining systems found in vertebrates (genotypic or GSD and temperature-dependent or TSD) requires a comprehensive and integrative examination from both a functional and an evolutionary perspective. Particularly revealing is the examination of the gene network that regulates gonadogenesis. Here, I review some advances in this field and propose some additional hypotheses about the composition of the gene network underlying sexual development, the functional links among some of its elements and their evolution in turtles. I focus on several pending questions about: (1) What renders TSD systems thermo-sensitive? (2) Is there one developmentally conserved or multiple TSD mechanisms? (3) Have evolutionarily derived GSD species lost all ancestral thermal-sensitivity? New data are presented on embryonic expression of Dax1 (the dosage-sensitive sex-reversal adrenal hypoplasia congenital on the X chromosome gene in the turtles Chrysemys picta (TSD) and Apalone mutica (GSD). No differential Dax1 expression was detected in C. picta at any of the stages examined, consistent with reports on two other TSD turtles and alligators. Notably, significantly higher Dax1 expression was found at 30°C than at 25°C at stage 15 in A. mutica (GSD), likely caused by Wt1's identical expression pattern previously reported. Because Sf1 is an immediate downstream target of Dax1 and its expression is not affected by temperature, it is proposed that Sf1 renders Dax1's differential signal ineffective to induce biased sex ratios in A. mutica, as previously proposed for Wt1's thermosensitive expression. Thus, it is hypothesized that Sf1 plays a major role in the lack of response of sex ratio to temperature of A. mutica, and may function as a sex-determining gene in this GSD species. These and previous data permit formulating several mechanistic hypotheses: (1) the postulation of Wt1 as a candidate thermal master switch alone, or in combination with Sf1, in the TSD turtle C. picta; (2) the proposition of Sf1 as a sex-determining gene in the GSD turtle A. mutica; and (3) the hypothesis that differing patterns of gene expression among TSD taxa reflect multiple traits from a developmental perspective. Moreover, the recent finding of relic differential Wt1 expression in A. mutica and the results for Dax1 in this species provide empirical evidence that GSD taxa can harbor thermal sensitivity at the level of gene expression, potentially co-optable during TSD evolution.

Stem cell-specific expression of Dax1 is conferred by STAT3 and Oct3/4 in embryonic stem cells

Embryonic stem (ES) cells are pluripotent cells derived from inner cell mass of blastocysts. An orphan nuclear receptor, Dax1, is specifically expressed in undifferentiated ES cells and plays an important role in their self-renewal. The regulatory mechanism of Dax1 expression in ES cells, however, remains unknown. In this study, we found that STAT3 and Oct3/4, essential transcription factors for ES cell self-renewal, are involved in the regulation of Dax1 expression. Suppression of either STAT3 or Oct3/4 resulted in down-regulation of Dax1. Reporter assay identified putative binding sites for these factors in the promoter/enhancer region of the Dax1 gene. Chromatin immunoprecipitation analysis suggested the in vivo association of STAT3 and Oct3/4 with the putative sites. Furthermore, gel shift assay indicated that these transcription factors directly bind to their putative binding sites. These results suggest that STAT3 and Oct3/4 control the expression of Dax1 to maintain the self-renewal of ES cells.

Chrysin, a natural flavonoid enhances steroidogenesis and steroidogenic acute regulatory protein gene expression in mouse Leydig cells

During the aging process of males, testosterone biosynthesis declines in testicular Leydig cells resulting in decreases in various physiological functions. To explore the possibility of delaying the decline using food supplements, we have studied steroidogenic effects of a natural flavonoid, chrysin, in mouse Leydig cells. Chrysin dramatically increased cyclic AMP (cAMP)-induced steroidogenesis in MA-10 mouse Leydig tumor cells. This result was confirmed using Leydig cells isolated from mouse testes. The steroidogenic effect of chrysin is not associated with an increase in expression of the P450 side-chain cleavage enzyme, required for the conversion of cholesterol to pregnenolone. In addition, when 22(R)hydroxylcholesterol was used as a substrate, chrysin induced a non-significant increase in steroid hormone, suggesting that the majority of the observed increase in steroidogenesis was due to the increased supply of substrate cholesterol. These observations were corroborated by showing that chrysin induced a marked increase in the expression of steroidogenic acute regulatory (StAR) protein, the factor that controls mitochondrial cholesterol transfer. Also, chrysin significantly increased StAR promoter activity and StAR mRNA level. Further studies indicated that this compound depressed expression of DAX-1, a repressor in StAR gene transcription. In the absence of cAMP, chrysin did not increase steroidogenesis. However, when a sub-threshold level of cAMP was used, StAR protein and steroid hormone were increased by chrysin to the levels seen with maximal stimulation of cAMP. These results suggest that while chrysin itself is unable to induce StAR gene expression and steroidogenesis, it appears to function by increasing the sensitivity of Leydig cells to cAMP stimulation.

Effect of &lt;I&gt;Sry&lt;/I&gt;silencing by siRNA on the expression of sex determining genes in mouse embryos

In order to investigate Sry regulation network in the development of male embryo, we inhibited the Sry gene expression by RNAi and then examined expression of other sex-related genes. Six genes (Sox9, Wt1, Sf1, Dax1, Gata4 and Amh), which are suggested to be closely related to Sry regulation were studied. Two siRNA expression vectors pSilencer4.1/Sry217 and pSilencer4.1/Sry565 were constructed and injected into gestated mouse through tail vein at 9.5 day of conception (dpc). The inhibition efficiency of Sry and the expression of other six genes were examined in male embryos at 11.5 dpc by RT-PCR and Western-blot. Expressions of the other six genes were analyzed by fluorescence quantity PCR. The results indicated both the pSilencer4.1/Sry217 and pSilencer4.1/Sry565 could inhibit significantly increased after Sry silencing. In contrast, no significant changes were observed in the expression of Sf1, Amh, Gata4, Dax1 and Sox9 when silencing Sry by siRNA. Our results suggested that the Wt1 transcription was regulated by Sry, whereas the Sox9 expression is not directly regulated by Sry in the development of genital ridge.

ウシ黄体内におけるステロイド産生転写調節因子であるDAX-1，SF-1及びLRH-1のmRNA発現変動

ウシ黄体内におけるステロイド産生転写調節因子であるDAX-1，SF-1及びLRH-1のmRNA発現変動

GERM CELL NUCLEAR FACTOR (GCNF) REPRESSES THE OCT4 GENE DURING RETINOIC ACID-INDUCED HUMAN EMBRYONIC STEM CELL DIFFERENTIATION

Embryonic stem (ES) cells require a network of transcription factors and signaling molecules to maintain their pluripotency. The transcription factor Oct4, one of pluripotency genes, is required for self-renewal and maintains the undifferentiated state of ES cells. Loss of expression of Oct4 initiates ES cell differentiation. Germ cell nuclear factor (GCNF), an orphan nuclear receptor, binds to a DR0 site within Oct4 gene promoter in human ES cells. Retinoic acid induces expression of GCNF, which binds the promoter of Oct4 gene, and accelerates the decrease of Oct4 gene expression and human ES cells differentiation. Expression of other pluripotency genes, such as Sox2, also decrease during human ES cell differentiation induced with retinoic acid. These findings increase our understanding of the mechanism of regulation of gene expression and in maintaining human ES cell pluripotence and during the differentiation process. Results 1: Tera-2 cells and human ES (H9) cells were induced to differentiate by Retinoic Acid (1μM). Expression of GCNF, Oct4, Sox2, Dax1, LRH1, SF1, Actin was determined by RT-PCR (A), and by western blot (B) in Tera-2 cells and human ES cells. GCNF expression increased by day 2 of differentiation in Tera-2 cells and human ES cells when treated with Retinoic Acid (1μM). GCNF expression is transient and decreased earlier in Tera-2 cells than in human ES cells. Expression of Oct4, Sox2 and SF-1 was gradually repressed during differentiation. Expression of the orphan receptor LRH1 increased when Tera-2 and human ES cells differentiated, and subsequently decreased. DAX1 expression was up-regulated when Tera-2 cells and human ES cells differentiate, and maintained the same level until day 6 in Tera-2 cells but gradually increased in human ES cells. Result 2: GCNF binding the DR0 site within the Oct4 promoter in Tera-2 cells and human ES cells is induced upon differentiation and is transient in nature. Detection of GCNF binding to the Oct4 promoter in vivo using Chromatin immunoprecipitation (ChIP) assays: Tera-2 cells and human ES cells were treated with Retinoic Acid (1 μM) and induced to differentiate. GCNF binding to the DR0 site of Oct4 promoter can be detected in vivo when Tera-2 cell and human ES cells differentiate, and then decreases at later stages of differentiation. Result 3: Analysis of GCNF binding to the DR0 element in the Oct4 promoter by electrophoretic mobility shift assays (EMSAs). No GCNF binding to the DR0 element was detected at day 0 in the Tera-2 cells and human ES cells. GCNF has been at the highest level of binding to the DR0 site at day 3 of RA-induced differentiation (arrow) and is shifted (arrow head) when GCNF-specific antibody is added to the EMSAs. GCNF DNA binding activity disappeared by day 5 of differentiation in Tera-2 cells. In human ES cells, GCNF displayed a higher level of binding to the DR0 site at day 3 of differentiation and decreased at day 5 of RA treatment. This result shows that GCNF directly binds to the conserved DR0 element in the Oct4 promoter in Tera-2 cells and human ES cell. (platform)

Developmental expression of DAX1 in the European sea bass, Dicentrarchus labrax: lack of evidence for sexual dimorphism during sex differentiation

BackgroundDAX1 (NR0B1), a member of the nuclear receptors super family, has been shown to be involved in the genetic sex determination and in gonadal differentiation in several vertebrate species. In the aquaculture fish European sea bass, Dicentrarchus labrax, and in the generality of fish species, the mechanisms of sex determination and differentiation have not been elucidated. The present study aimed at characterizing the European DAX1 gene and its developmental expression at the mRNA level.MethodsA full length European sea bass DAX1 cDNA (sbDAX1) was isolated by screening a testis cDNA library. The structure of the DAX1 gene was determined by PCR and Southern blot. Multisequence alignments and phylogenetic analysis were used to compare the translated sbDAX1 product to that of other vertebrates. sbDAX1 expression was analysed by Northern blot and relative RT-PCR in adult tissues. Developmental expression of mRNA levels was analysed in groups of larvae grown either at 15°C or 20°C (masculinising temperature) during the first 60 days, or two groups of fish selected for fast (mostly females) and slow growth.ResultsThe sbDAX1 is expressed as a single transcript in testis and ovary encoding a predicted protein of 301 amino acids. A polyglutamine stretch of variable length in different DAX1 proteins is present in the DNA binding domain. The sbDAX1 gene is composed of two exons, separated by a single 283 bp intron with conserved splice sites in same region of the ligand binding domain as other DAX1 genes. sbDAX1 mRNA is not restricted to the brain-pituitary-gonadal axis and is also detected in the gut, heart, gills, muscle and kidney. sbDAX1 mRNA was detected as early as 4 days post hatching (dph) and expression was not affected by incubation temperature. Throughout gonadal sex differentiation (60–300 dph) no dimorphic pattern of expression was observed.ConclusionThe sbDAX1 gene and putative protein coding region is highly conserved and has a wide pattern of tissue expression. Although gene expression data suggests sbDAX1 to be important for the development and differentiation of the gonads, it is apparently not sex specific.

Male-to-female sex reversal associated with an ∼250 kb deletion upstream of NR0B1 (DAX1)

Deletion of the dosage sensitive gene NR0B1 encoding DAX1 on chromosome Xp21.2 results in congenital adrenal hypoplasia (AHC), whereas NR0B1 duplication in 46,XY individuals leads to gonadal dysgenesis and a female phenotype. We describe a 21-year-old 46,XY female manifesting primary amenorrhea, a small immature uterus, gonadal dysgenesis, and notably absent adrenal insufficiency with a submicroscopic (257 kb) deletion upstream of NR0B1. We hypothesize that loss of regulatory sequences may have resulted in position effect up-regulation of DAX1 expression, consistent with phenotypic consequences of NR0B1 duplication. We propose that this genomic region and by extension those surrounding the dosage sensitive SRY, SOX9, SF1, and WNT-4 genes, should be examined for copy-number variation in patients with sex reversal.

Dax1 suppresses P450arom expression in medaka ovarian follicles

Dax1 is a member of an unusual orphan nuclear receptor family, and is known to regulate P450arom in mammals and is involved in sex differentiation in some vertebrates. To investigate whether Dax1 is involved in the regulation of the steroidogenic pathway for estrogen biosynthesis in medaka ovarian follicles, we isolated Dax1 cDNA from adult medaka ovaries and analyzed its expression pattern in medaka gonads. In adult ovaries, Dax1 mRNA was detected only in postvitellogenic follicles and was not detected in previtellogenic and vitellogenic follicles. In adult testis, Dax1 mRNA was not detected. We compared the expression pattern of Dax1 with that of Foxl2, Ad4BP/Sf-1, P450c17, and P450arom by in situ hybridization using adjacent sections. In contrast to Dax1 expression, these genes were co-expressed in vitellogenic follicles but were not detected in postvitellogenic follicles. Thus, in medaka ovarian follicles, Dax1 did not show any overlapping expression patterns against Foxl2, Ad4BP/Sf-1, P450c17, and P450arom. Moreover, co-transfection experiments demonstrated that Dax1 inhibits Ad4BP/Sf-1- and Foxl2-mediated P450arom expression. On the other hand, during early sex differentiation, Dax1 mRNA was not detected in both males and females. Our results suggest that Dax1 down-regulates Ad4BP/Sf-1- and Foxl2-mediated P450arom expression in medaka ovarian follicles.

An Amino-Terminal DAX1 (NROB1) Missense Mutation Associated with Isolated Mineralocorticoid Deficiency

Mutations in DAX1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1; NR0B1) cause X-linked adrenal hypoplasia congenita, a disease characterized by primary adrenal failure, testicular dysgenesis, and gonadotropin deficiency. Most DAX1 mutations are deletions, nonsense, or frameshift mutations that markedly impair its transcriptional activity. Missense mutations have been restricted to the carboxy-terminal domain and are associated with more variable clinical phenotypes. The objective was to identify novel clinical phenotypes associated with DAX1 missense mutations. We investigated the genetic basis of isolated mineralocorticoid deficiency in a patient who carries a unique missense mutation (W105C) in the amino-terminal region of DAX1. The W105C DAX1 mutation in the proband was present in three asymptomatic hemizygous males, but it was not detected in the general population. Using in vitro studies of DAX1 expression and function in transfected cells, we demonstrate that the mutant DAX1 protein exhibits mild loss of function, whether studied for genes it represses or for genes it activates. Structure-function studies suggest that the W105C and other mutations in the aminoterminus are compensated by the presence of repeated LXXLL motifs that mediate DAX1 interactions with other proteins. We describe the first missense mutation in the aminoterminus of DAX1 and conclude that mutations in this region may be partially compensated by redundant functional domains. Mild DAX1 mutations may be a cause of isolated mineralocorticoid deficiency.

Serum-free cultures of murine adrenal cortical cells

Purpose The feasibility of culturing murine adrenal cortical cells before transplantation was investigated in this study. Method Primary murine adrenal cortical cells were maintained in either fetal bovine and horse sera–containing media or serum-free media. Real-time polymerase chain reaction was used to quantify the levels of adrenal cortical gene expression in the cultured cells. Results The use of sera-containing media led to the growth of many cells in the culture, but the expression of Sf-1, Dax-1, and Cyp11b1 in such cultures declined rapidly. In contrast, there was no significant cell growth in the serum-free culture medium. Culturing murine adrenal cortical cells in the serum-free medium resulted in higher levels of Sf-1, Dax-1, and Cyp11b1 gene expression. In the serum-free medium, adrenal cortical cells also responded to adrenocorticotropic hormone by increasing the expression of Cyp11b1 and suppressing the expression of Dax-1 in a dose-dependent manner. The addition of basic fibroblast growth factor to the serum-free medium maintained the expression of Sf-1, Dax-1, and Cyp11b1 for 4 weeks. Conclusion Adrenal cortical cells isolated from adult mice were successfully maintained in a serum-free culture medium with basic fibroblast growth factor. This culture system may be suitable for further manipulation of adrenal cortical cells in vitro before transplantation.

In vivo inhibition of steroidogenic acute regulatory protein expression by dexamethasone parallels induction of the negative transcription factor DAX-1

In this study, we investigated the effect of dexamethasone on the synthesis of steroidogenic acute regulatory protein (StAR) and the expression of DAX-1 (dosage sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1) and SF-1 (steroidogenic factor-1) in vivo. Male rats were treated with dexamethasone (0.4 and 4 mg/kg body wt per day) by intraperitoneal injections using phosphate-buffered saline as the vehicle for 7 d. At the end of 7 d, serum testosterone levels were decreased. Response to luteinizing hormone (LH) and 8-bromo-cyclic-AMP (8-Br-cAMP) in vitro was reduced in testicular cells isolated from dexamethasone-treated rat testes. Dexamethasone decreased LH-stimulated cAMP production. The conversion of 22(R)-hydroxycholesterol, pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone, and androstenedione to testosterone was not affected by dexamethasone. Dexamethasone increased DAX-1 expression and concordantly decreased StAR protein and mRNA in testicular cells. The increase in DAX-1 protein corresponded to a 57% reduction in StAR mRNA levels concomitant with a 79% reduction in serum testosterone levels. Dexamethasone had no effect on the level of SF-1, but increased the amount of complexed DAX-1-SF-1. Dexamethasone in vitro suppressed StAR promoter activity when an increasing amount of DAX-1 cDNA was transfected. These results demonstrate that dexamethasone increases expression of DAX-1, which results in increased amounts of complexed DAX-1-SF-1, in the absence of any change in the expression of SF-1. These observations strongly support the concept that dexamethasone suppresses rat testicular testosterone production, at least in part, by increasing the amount of complexed DAX-1-SF-1 in these cells, which leads directly to decreased availability of free SF-1 and, therefore, decreased activation of transcription of the rat StAR gene.

Reciprocal Regulation of a Glucocorticoid Receptor-Steroidogenic Factor-1 Transcription Complex on the Dax-1 Promoter by Glucocorticoids and Adrenocorticotropic Hormone in the Adrenal Cortex

Numerous genes required for adrenocortical steroidogenesis are activated by the nuclear hormone receptor steroidogenic factor 1 (SF-1) (NR5A1). Dax-1 (NR0B1), another nuclear hormone receptor, represses SF-1-dependent activation. Glucocorticoid products of the adrenal cortex provide negative feedback to the production of hypothalamic CRH and pituitary ACTH. We hypothesized that glucocorticoids stimulate an intraadrenal negative feedback loop via activation of Dax-1 expression. Reporter constructs show glucocorticoid-dependent synergy between SF-1 and glucocorticoid receptor (GR) in the activation of Dax-1, which is antagonized by ACTH signaling. We map the functional glucocorticoid response element between -718 and -704 bp, required for activation by GR and synergy with SF-1. Of three SF-1 response elements, only the -128-bp SF-1 response element is required for synergy with GR. Chromatin immunoprecipitation (ChIP) assays demonstrate that dexamethasone treatment increases GR and SF-1 binding to the endogenous murine Dax-1 promoter 10- and 3.5-fold over baseline. Serial ChIP assays reveal that that GR and SF-1 are part of the same complex on the Dax-1 promoter, whereas coimmunoprecipitation assay confirms the presence of a protein complex that contains both GR and SF-1. ACTH stimulation disrupts the formation of this complex by abrogating SF-1 binding to the Dax-1 promoter, while promoting SF-1 binding to the melanocortin-2 receptor (Mc2r) and steroidogenic acute regulatory protein (StAR) promoters. Finally, dexamethasone treatment increases endogenous Dax-1 expression and concordantly decreases StAR expression. ACTH signaling antagonizes the increase in Dax-1 yet strongly activates StAR transcription. These data indicate that GR provides feedback regulation of adrenocortical steroid production through synergistic activation of Dax-1 with SF-1, which is antagonized by ACTH activation of the adrenal cortex.