Timing of adrenal regression controlled by synergistic interaction between Sf1 SUMOylation and Dax1.

The nuclear receptor steroidogenic factor 1 (Sf1, Nr5a1, Ad4bp) is crucial for formation, development and function of steroidogenic tissues. A fetal adrenal enhancer (FAdE) in the Sf1 gene was previously identified to direct Sf1 expression exclusively in the fetal adrenal cortex and is bound by both Sf1 and Dax1. Here, we have examined the function of Sf1 SUMOylation and its interaction with Dax1 on FAdE function. A diffused prolonged pattern of FAdE expression and delayed regression of the postnatal fetal cortex (X-zone) were detected in both the SUMOylation-deficient-Sf12KR/2KR and Dax1 knockout mouse lines, with FAdE expression/activity retained in the postnatal 20αHSD-positive postnatal X-zone cells. In vitro studies indicated that Sf1 SUMOylation, although not directly influencing DNA binding, actually increased binding of Dax1 to Sf1 to further enhance transcriptional repression of FAdE. Taken together, these studies define a crucial repressor function of Sf1 SUMOylation and Dax1 in the physiological cessation of FAdE-mediated Sf1 expression and the resultant regression of the postnatal fetal cortex (X-zone).

DAX-1 inhibits hepatocellular carcinoma proliferation by inhibiting ß-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 ß-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 ß-Catenin and attenuate its transcriptional activity. CONCLUSION: Therefore, our results suggest a previously unknown DAX-1/ß-Catenin molecular network controlling HCC development.

Are human male patients with DAX1/NR0B1 mutations infertile?

DAX-1 stands for Dosage sensitive sex-reversal, Adrenal hypoplasia congenital (AHC), on the X chromosome. DAX-1 mutations usually cause primary adrenal insufficiency or congenital adrenal hypoplasia in early childhood and hypogonadotropic hypogonadism (MIM # 300200). DAX-1 protein is necessary to maintain normal spermatogenesis. In humans, male fertility has been studied in few patients carrying DAX-1 mutations. Cases of azoospermia have been reported, as well as unsuccessful gonadotropin treatments. The clinician should be informed that TESE-ICSI technique carries a potential hope to father non-affected children, as shown in this review.

Functional effects of DAX-1 mutations identified in patients with X-linked adrenal hypoplasia congenita.

X-linked adrenal hypoplasia congenita with hypogonadotropic hypogonadism and adrenal insufficiency is a rare disorder caused by mutations of DAX-1. In this study, we investigated the functional defects of DAX-1 caused by mutations identified in 3 unrelated Korean patients with adrenal hypoplasia congenita. The DAX-1 gene was directly sequenced using genomic DNA isolated from peripheral blood leukocytes. The functional defects of DAX-1 caused by mutations were evaluated using an in vitro promoter assay. After mutagenesis of DAX-1 complementary DNA in the pcDNA3.1 vector, steroidogenic factor 1 and the promoter region of steroidogenic acute regulatory protein (StAR) genes in pGL4.10[luc2] were transiently cotransfected into human embryonic kidney 293 cells, followed by luminometry measurements of the luciferase activity of StAR. Mutation analysis of 3 patients revealed p.L386delfsX2, p.W105X, and p.Q252X mutations of the DAX-1 gene. The mutant DAX-1 proteins showed lower repressive activity on the StAR gene promoter when compared with normal DAX-1. Nonsense and frameshift mutations of the DAX-1 gene partially eliminated the ability of DAX-1 to repress the transcription of StAR in an in vitro assay.

Role of DAX-1 (NR0B1) and steroidogenic factor-1 (NR5A1) in human adrenal function.

The nuclear receptor transcription factors DAX-1 (NR0B1) and SF-1 (NR5A1) regulate many aspects of adrenal and reproductive development and function. Disruption of the genes encoding these factors can be associated with pediatric adrenal disease. DAX-1 mutations are classically associated with X-linked adrenal hypoplasia congenita, hypogonadotropic hypogonadism and impaired spermatogenesis. However, other phenotypes are also being reported, such as isolated mineralocorticoid insufficiency, premature sexual development, primary adrenal insufficiency in a 46, XX patient and late-onset X-linked adrenal hypoplasia congenita and/or hypogonadotropic hypogonadism. SF-1 mutations have also been associated with primary adrenal insufficiency, together with 46, XY disorders of sex development. However it is emerging that SF-1 changes are a relatively rare cause of primary adrenal failure in humans, and most individuals with SF-1 mutations have a spectrum of 46, XY disorders of sex development phenotypes. These conditions range from 46, XY females with streak gonads and müllerian structures, through children with ambiguous genitalia and inguinal testes, to severe penoscrotal hypospadias with undescended testes. Therefore, the human gonad appears to be more sensitive than the adrenal gland to loss of SF-1 function. This review will focus on the expanding range of phenotypes associated with DAX-1 and SF-1 mutations.

Targeting DAX-1 in embryonic stem cells and cancer.

IMPORTANCE OF THE FIELD: DAX-1 (NR0B1) is an unusual orphan nuclear receptor whose function is essential for the development of the human adrenal cortex and onset of puberty. Recent data have implicated this transcription factor also in embryonic stem cell and cancer biology. AREAS COVERED IN THIS REVIEW: The role of DAX-1 in the regulation of development and function of the adrenal cortex, reproductive axis, embryonic stem cells and a few types of cancer. WHAT THE READER WILL GAIN: Here we review the past and present milestones in DAX-1 research and try to provide hints about the development and fields of application of DAX-1-targeted drugs in the future. TAKE HOME MESSAGE: The unusual structure and restricted expression pattern of DAX-1 may offer unique opportunities for drug discovery.

Seven novel DAX1 mutations with loss of function identified in Chinese patients with congenital adrenal hypoplasia.

CONTEXT: 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. OBJECTIVE: The present study aimed at identifying DAX1 mutations in Chinese AHC patients and investigating the functional defects of detected novel mutations. PATIENTS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

PPAR-gamma coactivator-1alpha regulates progesterone production in ovarian granulosa cells with SF-1 and LRH-1.

Previously, we demonstrated that bone marrow-derived mesenchymal stem cells (MSCs) differentiate into steroidogenic cells such as Leydig and adrenocortical cells by the introduction of steroidogenic factor-1 (SF-1) and treatment with cAMP. In this study, we employed the same approach to differentiate umbilical cord blood (UCB)-derived MSCs. Despite UCB-MSCs differentiating into steroidogenic cells, they exhibited characteristics of granulosa-luteal-like cells. We found that peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) was expressed and further induced by cAMP stimulation in UCB-MSCs. Consistent with these results, tissue-specific expression of Pgc-1alpha was observed in rat ovarian granulosa cells. PGC-1alpha binds to the NR5A family [SF-1 and liver receptor homolog-1 (LRH-1)] of proteins and markedly enhances their transcriptional activities. Reporter assays revealed that PGC-1alpha activated the promoter activities of SF-1 and LRH-1 target genes. Infection of KGN cells (a human cell line derived from granulosa cells) with adenoviruses expressing PGC-1alpha resulted in the induction of steroidogenesis-related genes and stimulation of progesterone production. PGC-1alpha also induced SF-1 and LRH-1, with the latter induced to a greater extent. Knockdown of Pgc-1alpha in cultured rat granulosa cells resulted in attenuation of gene expression as well as progesterone production. Transactivation of the NR5A family by PGC-1alpha was repressed by Dax-1. PGC-1alpha binds to the activation function 2 domain of NR5A proteins via its consensus LXXLL motif. These results indicate that PGC-1alpha is involved in progesterone production in ovarian granulosa cells by potentiating transcriptional activities of the NR5A family proteins.

Control of sex development.

The process of sexual differentiation is central for reproduction of almost all metazoan, and therefore, for maintenance of practically all multicellular organisms. In sex development, we can distinguish two different processes, sex determination, that is the developmental decision that directs the undifferentiated embryo into a sexually dimorphic individual. In mammals, sex determination equals gonadal development. The second process known as sex differentiation takes place once the sex determination decision has been made through factors produced by the gonads that determine the development of the phenotypic sex. Most of the knowledge on the factors involved in sexual development came from animal models and from studies of cases in whom the genetic or the gonadal sex does not match the phenotypical sex, that is, patients affected by disorders of sex development (DSDs). Generally speaking, factors influencing sex determination are transcriptional regulators, whereas factors important for sex differentiation are secreted hormones and their receptors. This review focuses on these factors and whenever possible, references regarding the 'prismatic' clinical cases are given.