The orphan nuclear receptor steroidogenic factor 1 (SF-1, also called Ad4BP and officially designated NR5A1) has emerged as an essential regulator of endocrine development and function. Initially identified as a tissue-specific transcriptional regulator of the cytochrome P450 steroid hydroxylases, SF-1 has considerably broader roles, as evidenced from studies in knockout mice lacking SF-1. The SF-1-knockout mice lacked adrenal glands and gonads and therefore died from adrenal insufficiency within the first week after birth. In addition, SF-1 knockout mice exhibited male-to-female sex reversal of their internal and external genitalia, impaired expression of multiple markers of pituitary gonadotropes, and agenesis of the ventromedial hypothalamic nucleus (VMH). These studies delineated essential roles of SF-1 in regulating endocrine differentiation and function at multiple levels, particularly with respect to reproduction. This chapter will review the experiments that established SF-1 as a pivotal, global determinant of endocrine differentiation and function. We next discuss recent insights into the mechanisms controlling the expression and function of SF-1 as well as the current status of research aimed at delineating its roles in specific tissues. Finally, we highlight areas where additional studies are needed to expand our understanding of SF-1 action. I. Initial Isolation of Steroidogenic Factor 1Steroid hormones are essential for fluid and electrolyte balance, intermediary metabolism, sexual differentiation, and reproductive function. Once the pathways of steroid hormone biosynthesis were defined and shown to involve the concerted actions of several cytochrome P450 mixed-function oxidases, attention turned to elucidating the mechanisms that regulate the expression of these enzymes. With the isolation of the bovine 21-hydroxylase cDNA (White et al., 1984b), followed shortly thereafter by the cloning of cDNAs encoding the side-chain cleavage enzyme (Matteson et al., 1984;Morohashi et al., 1984) and 11-hydroxylase (John et al., 1984), these questions could be addressed at a molecular level.
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.
ACTH-dependent transcriptional activation of the bovine CYP17 gene (the gene encoding cytochrome P450 steroid 17 alpha-hydroxylase) involves two cAMP-responsive sequences (CRS1 and CRS2) located in the promoter region. Here we demonstrate that two nuclear orphan receptors, chicken ovalbumin upstream promoter transcription factor (COUP-TF) and steroidogenic factor-1 (SF-1), bind to the part of the CRS2 element that contains the repeated sequences AAGTCA and AGGTCA spaced by six nucleotides (repCRS2). Overexpression of COUP-TF and SF-1 in both steroidogenic and nonsteroidogenic cells demonstrated that SF-1 is an activator of repCRS2-dependent transcription of reporter genes. Furthermore, the SF-1-dependent transcription could be further stimulated by activation of the cAMP-dependent protein kinase. In contrast, COUP-TF alone had no effect on repCRS2-dependent reporter gene activity. Mutations that interfere with the binding of SF-1 to repCRS2 in vitro abolished the cAMP-induced activities mediated by the element in transfected Y1 cells. The mutational analysis of repCRS2 further indicated that the binding sites for the two receptors overlap, and electrophoretic mobility shift assays demonstrated that the receptors bound in a mutually exclusive manner. Overexpression of both SF-1 and COUP-TFI simultaneously demonstrated that COUP-TFI inhibited SF-1-dependent activation of reporter genes. Transient transfection experiments with a construct containing a -100/+19 base pair fragment from the bovine CYP17 gene demonstrated that SF-1 and COUP-TF had similar effects on the intact promoter as on the repCRS2/reporter gene constructs. Our data suggest that the two orphan receptors bind in a mutually exclusive manner to repCRS2 and that SF-1 is involved in the activation and COUP-TF in the repression of repCRS2-dependent transcription.
AimMaintenance of the blood and extracellular volume requires tight control of endothelial macromolecule permeability, which is regulated by cAMP signalling. This study probes the role of the cAMP mediators rap guanine nucleotide exchange factor 3 and 4 (Epac1 and Epac2) for in vivo control of microvascular macromolecule permeability under basal conditions.MethodsEpac1−/− and Epac2−/− C57BL /6J mice were produced and compared with wild‐type mice for transvascular flux of radio‐labelled albumin in skin, adipose tissue, intestine, heart and skeletal muscle. The transvascular leakage was also studied by dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI) using the MRI contrast agent Gadomer‐17 as probe.ResultsEpac1−/− mice had constitutively increased transvascular macromolecule transport, indicating Epac1‐dependent restriction of baseline permeability. In addition, Epac1−/− mice showed little or no enhancement of vascular permeability in response to atrial natriuretic peptide (ANP), whether probed with labelled albumin or Gadomer‐17. Epac2−/− and wild‐type mice had similar basal and ANP‐stimulated clearances. Ultrastructure analysis revealed that Epac1−/− microvascular interendothelial junctions had constitutively less junctional complex.ConclusionEpac1 exerts a tonic inhibition of in vivo basal microvascular permeability. The loss of this tonic action increases baseline permeability, presumably by reducing the interendothelial permeability resistance. Part of the action of ANP to increase permeability in wild‐type microvessels may involve inhibition of the basal Epac1‐dependent activity.
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