SF-1 controls the expression of the scavenger gene akr1b7: in vitro and in vivo approaches.
ENDOCRINE RESEARCH(2009)
Abstract
EXPANDED ABSTRACTThe akr1b7/mvdp gene (aldo keto reductase 1B7/mouse vas deferens protein) encodes a tissue-specific aldose reductase expressed in the steroidogenic glands of different rodents and in the vas deferens solely in mouse. In the former tissues this enzyme acts as a scavenger of isocaproaldehyde derived from side chain cleavage of cholesterol during the first step of steroidogenesis. Akr1b7/mvdp is ACTH-responsive in the adrenal cortex (ZF) while androgen is the main inducer in the vas deferens. We have previously established that the −510/ +41 akr1-b7 promoter fragment is able to reproduce the endogenous gene zona fasciculata restricted, ACTH controlled expression, in transgenic mice adrenals. Here, we report that three response elements contained within this promoter (positions −102, −458, −503) are able to bind SF-1, the essential regulator of steroidogenesis, although the low affinity site at −503 retains some other specific proteins present in Y1 nuclear extracts. Mutation of the −102 site results in a lowering of the activity of the −510/ +41 promoter in Y1 cells, whereas mutation of the −458 site induces a reduction both in the global activity and forskolin sensitivity of the promoter. Interestingly, differential mutations of the −503 site nucleotides either induce an increase or a decrease in the basal and forskolin-induced activity.Then we raised the question of the physiological importance of the atypical SF-1 binding site at position −102 that was previously described to overlap a proximal androgen responsive element (AREp) by in vitro studies. In order to investigate the in vivo requirement for the −102 SFRE/AREp for adrenal expression and ACTH regulation, we generated transgenic mice harboring mutant contructs of the 1.8-kb promoter driving the CAT reporter. Adrenal expression in adult mice is impaired in all lines harboring the mutated −102 SFRE/AREp. Impaired gene expression in mice harboring the mutated −102 SFRE/AREp transgene is linked to impaired SF-1 binding and not to impaired AR binding since we showed that akr1b7 gene expression is not affected in the adrenals of Tfm mice which are naturally defective for AR. Interestingly, requirement for the −102 SFRE/AREp varies during development since the mutant and the wild type transgenes share similar expression pattern until postanatal day 15 and differ thereafter. Finally, the −102 SFRE is not essential for hormonal regulation since its mutation does not affect transgene responsiveness to dexamethasone or ACTH replacement.Thus our data reveal a new type of SF-1 binding element illustrated by the −102 SFRE of the akr1b7 gene that affects promoter strength in adult mice without precluding adrenal-targeting, hormonal control or early postnatal expression. Interestingly, as for akr1b7 gene multiple SFREs are often found within regulatory regions of SF-1 target genes. Genetic evidence from patients and mice with heterozygous mutations in SF-1 locus, indicate that this factor functions, in both species, in a dose-dependent manner. SF-1 acting as a quantitative regulator, one can hypothesize that the number (or the accessibility) of the different SFREs within a given gene will be determinant to fine tune when (development), where (tissue), and how (hormone responsiveness) it will be expressed. This is consistent with our data concerning developmental effects of the mutation of −102 SFRE and could explain why the mutation of one SF-1 binding site (−102) living intact two others at −458 and −503, impairs gene expression in transgenic mice from postnatal day 15 to adulthood without significantly affecting early postnatal developmental expression or ACTH responsiveness.
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Key words
scavenger geneakr1b7
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