Marit Bakke

Molecular aspects of steroidogenic factor 1 (SF-1)

Steroidogenic factor 1 (SF-1, also called Ad4BP and NR5A1) is a nuclear receptor with critical roles in steroidogenic tissues, as well as in the brain and pituitary. In particular, SF-1 has emerged as an essential regulator of adrenal and gonadal functions and development. In the last few years, our knowledge on SF-1 has increased considerably at all levels, from the gene to the protein, and on its specific roles in different physiological processes. In this review, we discuss the current understanding on SF-1 with focus on the parameters that control the transcriptional capacity of SF-1 and the mechanisms that ensure proper stage- and tissue-specific expression of the gene encoding SF-1.

Pituitary-specific knockout of steroidogenic factor 1.

Knockout mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1) revealed its essential roles at multiple levels of endocrine development and function. These SF-1 knockout mice lacked adrenal glands and gonads, thereby manifesting adrenal insufficiency and sex reversal of their internal and external genitalia. Their pituitary gonadotropes failed to express several markers of normal differentiated function, and they lacked a specific hypothalamic nucleus, the ventromedial hypothalamic nucleus (VMH). Using the Cre-loxP system, we generated mice whose gene encoding SF-1 was inactivated specifically in the anterior pituitary. These pituitary-specific SF-1 knockout mice were sterile and never matured sexually. Their gonads weighed only approximately 5% of the weight of wild-type gonads. SF-1 immunoreactivity was absent in the anterior pituitary but was unaffected in the adrenal cortex, validating the selectivity of the gene targeting strategy. Consistent with an important role of SF-1 in gonadotropes, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were markedly decreased in the pituitary-specific SF-1 knockout mice. The pituitary-specific SF-1 knockout mice are a novel genetic model of hypogonadotropic hypogonadism and establish essential roles of SF-1 in gonadotropin expression.

SF-1: a critical mediator of steroidogenesis.

Studies in knockout mice have established that the orphan nuclear receptor steroidogenic factor 1 (SF-1) plays essential roles in the development and function of the primary steroidogenic organs. These SF-1 knockout mice lacked adrenal glands and gonads, causing adrenocortical insufficiency and sex reversal of their internal and external genitalia. They also had impaired expression of pituitary gonadotropins and agenesis of the ventromedial hypothalamic nucleus (VMH), confirming roles of SF-1 at all three levels of the hypothalamic-pituitary-steroidogenic organ axis. Ongoing experiments are directed at developing methods to inactivate SF-1 in a tissue-specific manner.

Transcriptional regulation of the bovine CYP17 gene by cAMP

The transcription of steroid hydroxylase genes is controlled by ACTH and cAMP in the adrenal cortex. In most instances the regulation appears to rely on transcription factors traditionally not associated with cAMP-dependent gene expression. For the non-traditional factors it remains necessary to elucidate the coupling of increases in intracellular cAMP and cAMP-dependent protein kinase (PKA) activity to the function of these proteins. The bovine CYP17 gene, which encodes the steroid 17 alpha-hydroxylase, contains two discrete DNA elements within its promoter and upstream region (CRS1 and CRS2) that individually can confer cAMP responsiveness. The CRS1 element is a target for PKA signalling and for negative regulation via the protein kinase C signal transduction pathway. The homeodomain protein Pbx1 enhances CRS1-dependent transcription, but additional CRS1-binding proteins remain to be identified. Furthermore it is not known how PKA regulates the activity of Pbx1 or its possible binding partners. Closer to the promoter, the nuclear orphan receptors SF-1 and COUP-TF have overlapping binding sites in CRS2 and they bind in a mutually exclusive manner with very similar affinities; 8 and 10 nM, respectively. SF-1 stimulates whereas COUP-TF inhibits transcription from the bovine CYP17 promoter. Together, the data suggest that cAMP-dependent control of the amounts of the activator SF-1 vs. the repressor COUP-TF could influence CRS2-dependent transcription. In addition, PKA may influence the phosphorylation of SF-1, thus increasing its activity. In vitro, PKA will elicit phosphorylation of SF-1. However, although SF-1 can be immunoprecipitated from adrenocortical cells as a phosphroprotein, we have not been able to show cAMP-dependent increase in net phosphorylation in intact cells. More careful examination of individual phosphorylation sites in SF-1 may still reveal hormone- and cAMP-induced phosphorylation of SF-1.

Deoxyribonucleic Acid Methylation Controls Cell Type-Specific Expression of Steroidogenic Factor 1

Steroidogenic factor 1 (SF1) is expressed in a time- and cell-specific manner in the endocrine system. In this study we present evidence to support that methylation of CpG sites located in the proximal promoter of the gene encoding SF1 contributes to the restricted expression pattern of this nuclear receptor. DNA methylation analyses revealed a nearly perfect correlation between the methylation status of the proximal promoter and protein expression, such that it was hypomethylated in cells that express SF1 but hypermethylated in nonexpressing cells. Moreover, in vitro methylation of this region completely repressed reporter gene activity in transfected steroidogenic cells. Bisulfite sequencing of DNA from embryonic tissue demonstrated that the proximal promoter was unmethylated in the developing testis and ovary, whereas it was hypermethylated in tissues that do not express SF1. Together these results indicate that the DNA methylation pattern is established early in the embryo and stably inherited thereafter throughout development to confine SF1 expression to the appropriate tissues. Chromatin immunoprecipitation analyses revealed that the transcriptional activator upstream stimulatory factor 2 and RNA polymerase II were specifically recruited to this DNA region in cells in which the proximal promoter is hypomethylated, providing functional support for the fact that lack of methylation corresponds to a transcriptionally active gene. In conclusion, we identified a region within the SF1/Sf1 gene that epigenetically directs cell-specific expression of SF1.

Transcriptional regulation of the bovine CYP17 gene: Two nuclear orphan receptors determine activity of cAMP-responsive sequence 2

The CYP17 gene contains in its promoter region at least two cis-acting elements (cAMP-responsive sequence 1 and 2, CRS1 and CRS2) that are necessary for adrenocorticotropin (ACTH) induced transcription. The CRS2 element contains a 6 bp repeat similar to binding sites for members of the nuclear hormone receptor superfamily of transcription factors. We present data that establish the repeated part of CRS2 (repCRS2) as a target of two nuclear orphan receptors; steroidogenic factor 1 (SF-1) and chicken ovalbumin upstream promoter transcription factor (COUP-TF). The repCRS2 element was found to form COUP-TF-related complexes with nuclear extracts from all cell lines tested, whereas SF-1-related complexes were only formed with extracts from steroidogenic Y1 cells. Transfection studies of steroidogenic cells demonstrated that SF-1 acts as an activator of repCRS2-dependent transcription of reporter genes.

Development of a transgenic green fluorescent protein lineage marker for steroidogenic factor 1

Knockout (KO) mice lacking steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a complex phenotype that includes adrenal and gonadal agenesis, impaired function of pituitary gonadotropes, and abnormalities of the ventromedial hypothalamic nucleus (VMH). To develop a lineage marker for cells that express SF-1, we used bacterial artificial chromosome (BAC) transgenesis. A BAC fragment containing 50 kb of the mouse Nr5a1 gene was placed upstream of the coding sequence for enhanced green fluorescent protein (eGFP) and used to generate SF-1/eGFP transgenic mice. These sequences directed eGFP expression to multiple cell lineages that express SF-1, including steroidogenic cells of the adrenal cortex, testes, and ovaries, VMH neurons, and reticuloendothelial cells of the spleen. Despite the essential role of SF-1 in gonadotropes, eGFP was not expressed in the anterior pituitary. These studies show that 50 kb of the mouse Nr5a1 gene can target transgenic expression to multiple cell lineages that normally express SF-1. The SF-1/eGFP transgene provides a valuable tool to expand our understanding of the actions of SF-1 in endocrine development and function.

DNA Methylation of Alternative Promoters Directs Tissue Specific Expression of Epac2 Isoforms

Epac 1 and Epac 2 (Epac1/2; exchange factors directly activated by cAMP) are multidomain proteins that mediate cellular responses upon activation by the signaling molecule cAMP. Epac1 is ubiquitously expressed, whereas Epac2 exhibits a restricted expression pattern. The gene encoding Epac2 gives rise to at least three protein isoforms (Epac2A, Epac2B and Epac2C) that exhibit confined tissue and cell specific expression profiles. Here, we describe alternative promoter usage for the different isoforms of Epac2, and demonstrate that the activity of these promoters depend on the DNA methylation status. Bisulfite sequencing demonstrated that the level of methylation of the promoters in different tissues correlates with Epac2 isoform expression. The presented data indicate that the tissue-specific expression of the Epac2 isoforms is epigenetically regulated, and identify tissue-specific differentially methylated promoter regions within the Epac2 locus that are essential for its transcriptional control.

Approaches to define the role of SF-1 at different levels of the hypothalamic-pituitary-steroidogenic organ axis.

Targeted gene disruption has produced knockout mice globally deficient in the orphan nuclear receptor steroidogenic factor 1 (SF-1). These SF-1 knockout mice lacked adrenal glands and gonads and consequently exhibited adrenocortical insufficiency and sex reversal of their internal and external genitalia. They also had impaired expression of gonadtropins in the anterior pituitary gonadotropes and agenesis of the ventromedial hypothalamic nucleus (VMH), confirming roles of SF-1 at all three levels of the hypothalamic-pituitary-steroidogenic organ axis. Ongoing experiments are directed at using evolving techniques for tissue-specific gene inactivation to define the roles of SF-1 within discrete sites of the hypthalamic-pituitary-steroidogenic organ axis.

Mechanism behind Resistance against the Organophosphate Azamethiphos in Salmon Lice (Lepeophtheirus salmonis)

Acetylcholinesterase (AChE) is the primary target for organophosphates (OP). Several mutations have been reported in AChE to be associated with the reduced sensitivity against OP in various arthropods. However, to the best of our knowledge, no such reports are available for Lepeophtheirus salmonis. Hence, in the present study, we aimed to determine the association of AChE(s) gene(s) with resistance against OP. We screened the AChE genes (L. salmonis ace1a and ace1b) in two salmon lice populations: one sensitive (n=5) and the other resistant (n=5) for azamethiphos, a commonly used OP in salmon farming. The screening led to the identification of a missense mutation Phe362Tyr in L. salmonis ace1a, (corresponding to Phe331 in Torpedo californica AChE) in all the samples of the resistant population. We confirmed the potential role of the mutation, with reduced sensitivity against azamethiphos in L. salmonis, by screening for Phe362Tyr in 2 sensitive and 5 resistant strains. The significantly higher frequency of the mutant allele (362Tyr) in the resistant strains clearly indicated the possible association of Phe362Tyr mutation in L. salmonis ace1a with resistance towards azamethiphos. The 3D modelling, short term survival experiments and enzymatic assays further supported the imperative role of Phe362Tyr in reduced sensitivity of L. salmonis for azamethiphos. Based on all these observations, the present study, for the first time, presents the mechanism of resistance in L. salmonis against azamethiphos. In addition, we developed a rapid diagnostic tool for the high throughput screening of Phe362Tyr mutation using High Resolution Melt analysis.