Steroidogenic Factor 1, a Goldilocks Transcription Factor from Adrenocortical Organogenesis to Malignancy.

Steroidogenic factor-1 (SF-1, also termed Ad4BP; NR5A1 in the official nomenclature) is a nuclear receptor transcription factor that plays a crucial role in the regulation of adrenal and gonadal development, function and maintenance. In addition to its classical role in regulating the expression of P450 steroid hydroxylases and other steroidogenic genes, involvement in other key processes such as cell survival/proliferation and cytoskeleton dynamics have also been highlighted for SF-1. SF-1 has a restricted pattern of expression, being expressed along the hypothalamic-pituitary axis and in steroidogenic organs since the time of their establishment. Reduced SF-1 expression affects proper gonadal and adrenal organogenesis and function. On the other hand, SF-1 overexpression is found in adrenocortical carcinoma and represents a prognostic marker for patients' survival. This review is focused on the current knowledge about SF-1 and the crucial importance of its dosage for adrenal gland development and function, from its involvement in adrenal cortex formation to tumorigenesis. Overall, data converge towards SF-1 being a key player in the complex network of transcriptional regulation within the adrenal gland in a dosage-dependent manner.

Two pair-rule responsive enhancers regulate wingless transcription in the Drosophila blastoderm embryo.

BACKGROUND: While many developmentally relevant enhancers act in a modular fashion, there is growing evidence for nonadditive interactions between distinct cis-regulatory enhancers. We investigated if nonautonomous enhancer interactions underlie transcription regulation of the Drosophila segment polarity gene, wingless. RESULTS: We identified two wg enhancers active at the blastoderm stage: wg 3613u, located from -3.6 to -1.3 kb upstream of the wg transcription start site (TSS) and 3046d, located in intron two of the wg gene, from 3.0 to 4.6 kb downstream of the TSS. Genetic experiments confirm that Even Skipped (Eve), Fushi-tarazu (Ftz), Runt, Odd-paired (Opa), Odd-skipped (Odd), and Paired (Prd) contribute to spatially regulated wg expression. Interestingly, there are enhancer specific differences in response to the gain or loss of function of pair-rule gene activity. Although each element recapitulates aspects of wg expression, a composite reporter containing both enhancers more faithfully recapitulates wg regulation than would be predicted from the sum of their individual responses. CONCLUSION: These results suggest that the regulation of wg by pair-rule genes involves nonadditive interactions between distinct cis-regulatory enhancers.

Acute phenanthrene toxicity to juvenile diploid and triploid African catfish (Clarias gariepinus): Molecular, biochemical, and histopathological alterations.

Information on the biological responses of polyploid animals towards environmental contaminants is scarce. This study aimed to compare reproductive axis-related gene expressions in the brain, plasma biochemical responses, and the liver and gill histopathological alterations in diploid and triploid full-sibling juvenile African catfish (Clarias gariepinus). Fish were exposed for 96 h to one of the two waterborne phenanthrene (Phe) concentrations [mean measured (SD): 6.2 (2.4) and 76 (4.2) µg/L]. In triploids, exposure to 76 µg/L Phe increased mRNA level of fushi tarazu-factor 1 (ftz-f1). Expression of tryptophan hydroxylase2 (tph2) was also elevated in both ploidies following the exposure to 76 µg/L Phe compared to the solvent control. In triploids, 76 µg/L Phe increased plasma alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) levels compared to the other Phe-exposed group. It also elevated lactate and glucose contents relative to the other groups. In diploids, however, biochemical biomarkers did not change. Phenanthrene exposures elevated glycogen contents and the prevalence of histopathological lesions in the liver and gills of both ploidies. This study showed substantial differences between diploids and triploids on biochemical and molecular biomarker responses, but similar histopathological alterations following acute Phe exposures.

Analysis of Drosophila segmentation network identifies a JNK pathway factor overexpressed in kidney cancer.

We constructed a large-scale functional network model in Drosophila melanogaster built around two key transcription factors involved in the process of embryonic segmentation. Analysis of the model allowed the identification of a new role for the ubiquitin E3 ligase complex factor SPOP. In Drosophila, the gene encoding SPOP is a target of segmentation transcription factors. Drosophila SPOP mediates degradation of the Jun kinase phosphatase Puckered, thereby inducing tumor necrosis factor (TNF)/Eiger-dependent apoptosis. In humans, we found that SPOP plays a conserved role in TNF-mediated JNK signaling and was highly expressed in 99% of clear cell renal cell carcinomas (RCCs), the most prevalent form of kidney cancer. SPOP expression distinguished histological subtypes of RCC and facilitated identification of clear cell RCC as the primary tumor for metastatic lesions.

Dynactin suppresses the retrograde movement of apically localized mRNA in Drosophila blastoderm embryos.

Motor dependent transport of mRNA is a key mechanism in axis specification during development. Apical transport and anchoring of wingless and pair-rule transcripts in the Drosophila syncytial blastoderm embryo is mediated by cytoplasmic Dynein, the major minus end directed microtubule dependent molecular motor. Here, we show that, despite apical transport of mRNA being highly directional, mRNA particles often pause and move backward toward the plus ends of microtubules. We suggest that this retrograde movement helps overcome cellular obstructions. We show that the plus end movement of apical mRNA is independent of the major plus end microtubule motors Kinesin-1 and Kinesin-2. In contrast, Dynactin, a Dynein processivity factor, is required to suppress retrograde mRNA movements, as well as for efficient minus end motility. We propose that Dynein itself, rather than the activity of a plus end motor, is responsible for the plus end movements of the mRNA and that Dynactin is involved in preventing short reverse movements of the Dynein motor, known to occur in vitro.

Reproductive stage-related effects of salmon GnRH and sex steroid hormones on expression of genes encoding fushi tarazu factor 1 homolog and estrogen receptor alpha in masu salmon pituitary cells in vitro.

Expression of genes encoding gonadotropin (GTH) subunits in the salmon pituitary was regulated by salmon gonadotropin-releasing hormone (sGnRH) and sex steroid hormones in a reproductive stage-dependent manner, probably through DNA-binding transcription factors. Direct effects of these hormones on expression of genes encoding salmon fushi tarazu factor 1 homolog (sFF1-I) and estrogen receptor alpha (ERalpha) were therefore examined by use of primary pituitary cell cultures of masu salmon at different reproductive stages. Pituitaries were collected in March (before initiation of gonadal maturation), in May (early maturing), in July (late maturing), and in September (spawning period). Amounts of sFF1-I and ERalpha mRNAs in the pituitary cells were determined by real-time polymerase chain reactions after a treatment with sGnRH, estradiol-17beta (E2), testosterone (T) or 11-ketotestosterone (11KT). The amounts of sFF1-I mRNA were elevated by E2 in the males, and by sGnRH and T in the females before initiation of gonadal maturation and at the early maturing stage. The amounts of ERalpha mRNA in the early maturing females were elevated by sGnRH. Effects of sGnRH were not significant at the late maturing and spawning stages. The amounts of ERalpha mRNA in the spawning males were halved by 11KT and E2, and those of sFF1-I and ERalpha mRNAs in the late maturing females were decreased by T and 11KT. These results indicated that responsiveness of sFF1-I and ERalpha genes to sGnRH and sex steroid hormones is seasonally variable in relation to reproductive stages. Expression of sFF1 and ERalpha genes should be stimulated at the early stages of gonadal maturation prior to increases in the amounts of GTH subunit mRNAs, while attenuated after the late maturing period when stored amounts of GTH subunit mRNAs reached near the maximum.

The HMG-box protein Lilliputian is required for Runt-dependent activation of the pair-rule gene fushi-tarazu.

lilliputian (lilli), the sole Drosophila member of the FMR2/AF4 (Fragile X Mental Retardation/Acute Lymphoblastic Leukemia) family of transcription factors, is widely expressed with roles in segmentation, cellularization, and gastrulation during early embryogenesis with additional distinct roles at later stages of embryonic and postembryonic development. We identified lilli in a genetic screen based on the suppression of a lethal phenotype that is associated with ectopic expression of the transcription factor encoded by the segmentation gene runt in the blastoderm embryo. In contrast to other factors identified by this screen, lilli appears to have no role in mediating either the establishment or maintenance of engrailed (en) repression by Runt. Instead, we find that Lilli plays a critical role in the Runt-dependent activation of the pair-rule segmentation gene fushi-tarazu (ftz). The requirement for lilli is distinct from and temporally precedes the Runt-dependent activation of ftz that is mediated by the orphan nuclear receptor protein Ftz-F1. We further describe a role for lilli in the activation of Sex-lethal (Sxl), an early target of Runt in the sex determination pathway. However, lilli is not required for all targets that are activated by Runt and appears to have no role in activation of sloppy paired (slp1). Based on these results we suggest that Lilli plays an architectural role in facilitating transcriptional activation that depends both on the target gene and the developmental context.

TFIIH trafficking and its nuclear assembly during early Drosophila embryo development.

We present the first analysis of the dynamics of the transcription DNA-repair factor TFIIH at the onset of transcription in early Drosophila development. TFIIH is composed of ten polypeptides that are part of two complexes - the core and the CAK. We found that the TFIIH core is initially located in the cytoplasm of syncytial blastoderm embryos, and that after mitotic division ten and until the cellular blastoderm stage, the core moves from the cytoplasm to the nucleus. By contrast, the CAK complex is mostly cytoplasmic during cellularization and during gastrulation. However, both components are positioned at promoters of genes that are activated at transcription onset. Later in development, the CAK complex becomes mostly nuclear and co-localizes in most chromosomal regions with the TFIIH core, but not in all sites, suggesting that the CAK complex could have a TFIIH-independent role in transcription of some loci. We also demonstrate that even though the CAK and the core coexist in the early embryo cytoplasm, they do not interact until they are in the nucleus and suggest that the complete assembly of the ten subunits of TFIIH occurs in the nucleus at the mid-blastula transition. In addition, we present evidence that suggests that DNA helicase subunits XPB and XPD are assembled in the core when they are transported into the nucleus and are required for the onset of transcription.

Zebrafish sex determination and differentiation: involvement of FTZ-F1 genes.

Sex determination is the process deciding the sex of a developing embryo. This is usually determined genetically; however it is a delicate process, which in many cases can be influenced by environmental factors. The mechanisms controlling zebrafish sex determination and differentiation are not known. To date no sex linked genes have been identified in zebrafish and no sex chromosomes have been identified. However, a number of genes, as presented here, have been linked to the process of sex determination or differentiation in zebrafish. The zebrafish FTZ-F1 genes are of central interest as they are involved in regulating interrenal development and thereby steroid biosynthesis, as well as that they show expression patterns congruent with reproductive tissue differentiation and function. Zebrafish can be sex reversed by exposure to estrogens, suggesting that the estrogen levels are crucial during sex differentiation. The Cyp19 gene product aromatase converts testosterone into 17 beta-estradiol, and when inhibited leads to male to female sex reversal. FTZ-F1 genes are strongly linked to steroid biosynthesis and the regulatory region of Cyp19 contains binding sites for FTZ-F1 genes, further linking FTZ-F1 to this process. The role of FTZ-F1 and other candidates for zebrafish sex determination and differentiation is in focus of this review.

Insulin enhances the transcription of luteinizing hormone-beta gene.

OBJECTIVE: Hyperinsulinemia and insulin resistance are implicated in the pathophysiology of polycystic ovary syndrome, a condition associated with elevated levels of LH. We tested the hypothesis that insulin enhances the transcriptional activity of LHbeta promoter. STUDY DESIGN: We transfected the gonadotrope cell line LbetaT2 with a plasmid that expresses the proximal promoter of LHbeta gene upstream of luciferase, and determined insulin effect on endogenous LHbeta mRNA. RESULTS: We found that insulin stimulated (2-4 fold) the activity of this promoter in a time- and concentration-dependent manner. Accordingly, insulin up-regulated the level of LHbeta mRNA. In contrast, insulin had no significant effect on GnRH-dependent LHbeta expression. The expression of Egr-1 and SF-1, which are essential for transcription of LHbeta gene, was unchanged by insulin. CONCLUSION: Insulin enhances the transcription of LHbeta gene. This modulation may contribute to the pathophysiology of polycystic ovary syndrome.

Steroidogenic factor 1 differentially regulates basal and inducible steroidogenic gene expression and steroid synthesis in human adrenocortical H295R cells.

The significance of steroidogenic factor 1 (SF-1) in adrenal steroidogenesis was studied using adrenocortical cell lines transformed with a dominant negative mutant of SF-1. Constitutive expression of the mutant did not only impair the activity of endogenous SF-1 but also diminish its own expression, suggesting that SF-1 was under autoregulation. Inhibition of the endogenous SF-1 activity significantly reduced basal and inducible transcription of CYP17, CYP21B and CYP11B1, but exhibited little effects on StAR and CYP11A1 expression. Stimulating the transformed cells with potassium and cAMP freed CYP11B2 from the mutant-caused transcriptional inhibition, whereas the transformation abolished induction of CYP17 by both stimulants. Consistent with the transcriptional changes of steroidogenic genes, basal and inducible synthesis of cortisol and androgens drastically declined in the transformed cell lines. The relief of CYP11B2 repression following the potassium and cAMP stimulation removed the restraint the mutant exerted on aldosterone synthesis, and resulted in aldosterone overproduction in the stimulated transformed cells. SF-1 also plays a role in regulating the adrenocorticotrophic hormone (ACTH) responsiveness of the adrenocortical cells. Inhibition of SF-1 activity significantly decreased basal expression of ACTH receptor and its induction by potassium and cAMP.

Activation of protein kinase A alters subnuclear distribution pattern of human steroidogenic factor 1 in living cells.

BACKGROUND: The aim of this study was to identify the subnuclear distribution pattern of human orphan nuclear receptor steroidogenic factor 1 (SF-1) in living cells with and without the activation of protein kinase A (PKA) signal pathway, and thus try to explain the unknown mechanism by which PKA potentiates SF-1 transactivation. METHODS: Full-length cDNAs of wild type and a naturally occurring mutant (G35E) human SF-1 were cloned and fused with green fluorescent protein (GFP). Subcellular distribution pattern of human SF-1 in living cells, whose PKA signaling was either activated or not, was studied by laser confocal microscopy after the validity of the gene sequence was confirmed. RESULTS: The transactivation ability of the GFP-SF-1 chimeric protein was highly conserved. Wild type human SF-1 diffused homogeneously within the nuclei of cells when PKA was not active, and converged to clear foci when PKA was activated. Mutant SF-1 diffused within the nuclei even in the presence of PKA activation, surprisingly aggregating as fluorescent dots inside the nucleoli, a phenomenon not altered by PKA. CONCLUSIONS: Activation of PKA causes wild type, but not mutant SF-1 to alter its subnuclear distribution pattern to a transactivationally active form (foci formation). This finding may throw new light on the mechanism by which PKA activates the orphan nuclear receptor.

[Pathology of adrenal incidentaloma].

Adrenal incidentaloma or incidentally detected adrenal mass has recently increased in number because of the recent advancement of radiological diagnostic means. The clinical management and histopathological diagnosis of the resected adrenal mass has therefore become increasingly important. When surgical pathologists evaluate a resected adrenal mass of the patients with adrenal incidentaloma, it is very important to evaluate the following pathological aspects; 1. the mass is malignant or not? and 2. the mass is of adrenocortical origin or not. We will describe these aspects regarding the pathology of adrenal incidentaloma in this review with emphasis on the three aspects above.

[An ambiguous role of steroidogenic factor 1 in the rat GnRH receptor gene expression. Lessons from transgenic mice]. / Le rôle ambigu du facteur de transcription SF-1 dans l'expression génique du récepteur de la GnRH. Leçons de la transgenèse.

Because the GnRH receptor plays a paramount role within the reproductive axis, the understanding of the molecular apparatus that governs the tissue-specific expression and regulation of this gene must lead to a better knowledge of the physiology and the physiopathology of the gonadotrope function. To elucidate these mechanisms, we have used two complementary in vivo and in vitro approaches. Firstly, we have isolated the pituitary promoter of the rat GnRH receptor gene and investigated its activity using transient transfection into two gonadotrope-derived cell lines, the alphaT3-1 and the LbetaT2 cell lines. We have thus defined a primary set of transcription factors involved in the tissue-specific expression of the GnRH receptor gene. These include the steroidogenic factor-1 (SF-1) which plays a decisive role while functionally interacting with proteins related to the GATA and LIM homeodomain families of transcription factors. In addition, we highlighted the critical implication of SF-1 and its functional interaction with a CREB-related factor in the stimulatory action of PACAP (Pituitary Adenylate Cyclase Activating Polypeptide) on promoter activity. These results have led us to analyze the activity of this promoter by transgenesis in the mouse using human placental alkaline phosphatase as a reporter gene. In agreement with the in vitro data, the pituitary promoter was found to confer gonadotrope-specific activity in the pituitary. It was also able to direct transgene expression in several areas of the central nervous system known to express the endogenous GnRH receptor, in particular in the hippocampo-septal complex. Some of these tissue do not express SF-1, suggesting that, in vivo, its role would not be as decisive as suggested by the in vitro experiments. Surprisingly, during pituitary ontogenesis, the transgene is expressed as early as E 13.5 whereas SF-1 is not yet present in the pituitary. Thus, in vivo, SF-1 would not be necessary for the activation of the GnRH receptor gene during the early developmental stages in the pituitary. These results are consistent with data obtained following general or pituitary-specific knockout of the gene encoding SF-1, suggesting that the GnRH receptor is expressed despite the absence of this factor. Identifying the factors responsible for the activation of the GnRH receptor gene at these early developmental stages should make it possible to refine the role of SF-1, not only in gene regulation but more generally, in the physiology and the physiopathology of the gonadotrope function.

Characterization of binding between SF-1 and Sp1: predominant interaction of SF-1 with the N-terminal region of Sp1.

The transcription factor specificity protein 1 (Sp1) binds to GC boxes and interacts with many transcription factors to regulate gene expression. Steroidogenic factor-1 (SF-1) is an orphan nuclear receptor and plays a major role in regulation of the human steroidogenic acute regulatory (StAR) gene. We demonstrated that there is interaction between SF-1 and Sp1 on the human StAR promoter. In the present study, we examined the mechanism of the interaction between Sp1 and SF-1 on the human StAR gene promoter. Results of glutathione S-transferase (GST) pull-down assays and a mammalian two-hybrid assay showed that SF-1 interacted with Sp1 through the N-terminal domains of Sp1. Results of electrophoretic mobility shift assays using nuclear extracts showed that Sp1 is associated with SF-1-DNA complex formation. The density of SF-1-DNA complex was much greater when recombinant Sp1 was added to the incubation mixture. These results suggest that Sp1 interacts with SF-1 and that Sp1 enhances SF-1-DNA complex formation to regulate human StAR transcription.

Expression of aromatase and steroidogenic factor 1 in the lung of the urodele amphibian Pleurodeles waltl.

We report here the results of the analysis of aromatase and steroidogenic factor 1 (Sf1) expression in adult lung of the urodele amphibian Pleurodeles waltl. Using RT-PCR experiments, we show the expression of the estrogen-synthesizing enzyme, aromatase, in this organ. In the lung, no significant difference between males and females was observed in the level of aromatase mRNAs. Aromatase mRNA levels were also identical to those found in the brain or the testis, but the levels were 2-fold lower than in the ovary. Aromatase activity measurements revealed the presence of an active form of aromatase in the lung, which was similar in males and females. There was no difference in the level of aromatase activity between lung, brain, and testis, but a higher activity was measured in the ovary (13.7-fold compared with testis). Therefore, the differences in aromatase mRNA level between the ovary and the other organs did not mirror the differences in aromatase activity, suggesting the involvement of posttranslational events. Aromatase was also expressed in the lung of the anuran amphibian Xenopus laevis. In Pleurodeles lung, Sf1 mRNAs were also detected. There was no difference between males and females in the level of these mRNAs. The Sf1 mRNA levels were not significantly different from those measured in the brain, but a significant 2.1-fold higher level of expression was found in the gonads. These results demonstrate clearly the expression of steroidogenic markers in the adult lung of amphibians, but the biological significance of this remains to be determined.

Unique functional properties of a member of the Fushi Tarazu-Factor 1 family from Schistosoma mansoni.

SmFtz-F1 (Schistosoma mansoni Fushi Tarazu-Factor 1) belongs to the Ftz-F1 subfamily of nuclear receptors, but displays marked structural differences compared with its mammalian homologues SF-1 (steroidogenic factor-1) or liver receptor homologue-1. These include a long F domain (104 amino acids), an unusually large hinge region (133 amino acids) and a poorly conserved E-domain. Here, using Gal4 constructs and a mammalian two-hybrid assay, we have characterized the roles of these specific regions both in the transcriptional activity of the receptor and in its interactions with cofactors. Our results have shown that, although the AF-2 (activation function-2) region is the major activation function of the receptor, both the F and D domains are essential for AF-2-dependent activity. Modelling of SmFtz-F1 LBD (ligand-binding domain) and structure-guided mutagenesis allowed us to show the important role of helix H1 in maintaining the structural conformation of the LBD, and suggested that its autonomous transactivation activity, also observed with SF-1, is fortuitous. This strategy also allowed us to study an eventual ligand-dependence for this orphan receptor, the predicted three-dimensional models suggesting that the SmFtz-F1 LBD contains a large and well-defined ligand-binding pocket sealed by two arginine residues orientated towards the interior of the cavity. Mutation of these two residues provoked a loss of transcriptional activity of the receptor, and strongly reduced its interaction with SRC1 (steroid receptor cofactor-1), suggesting a ligand-dependent activity for SmFtz-F1. Taken together, our results argue for original and specific functional activities for this platyhelminth nuclear receptor.

[Differentiation and development of internal sexual organs, and müllerian inhibiting substance].

Internal sexual organs are differentiated and developed by androgens and regressed by müllerian inhibiting substance(anti-müllerian hormone). The role of 5 alpha-dihydrotestosterone, reduced form of testosterone by 5 alpha-reductase, in terms of development of Wolffian duct is discussed with soluble mesenchymal factor responsible for the epithelial branching morphogenesis of mouse seminal vesicle on the basis of experimental results using organ culture assay of mouse new-born seminal vesicle. An update of müllerian inhibiting substance, a fetal regressor of female internal organs such as uterus, fallopian tubes and upper third vagina, is also discussed.