Differential suppressive effects of low physiological doses of estradiol-17β in vivo on levels of mRNAs encoding steroidogenic acute regulatory protein and three steroidogenic enzymes in previtellogenic ovarian follicles of rainbow trout

Abstract LH triggers the biosynthesis of androgens in the theca-interstitial (T-I) cells of ovary through the activation of a cAMP-dependent pathway. We have previously shown that LH/human chorionic gonadotropin (hCG) activates mammalian target of rapamycin complex 1 (mTORC1) signaling network, leading to cell proliferation. In the present study, we provide evidence that the LH/hCG-mediated activation of the mTORC1 signaling cascade is involved in the regulation of steroidogenic enzymes in androgen biosynthesis. Treatment with LH/hCG increased the expression of downstream targets of mTORC1, ribosomal protein S6 kinase 1, and eukaryotic initiation factor 4E as well as steroidogenic enzymes. LH/hCG-mediated stimulation of the steroidogenic enzyme mRNA was blocked by the mTORC1 inhibitor, rapamycin. This inhibitory effect was selective because rapamycin failed to block hCG-mediated increase in the expression of Star mRNA levels. Furthermore, pharmacological targeting of mTORC1 with rapamycin also blocked LH/hCG- or forskolin-induced expression of cAMP response element-binding protein (CREB) and steroidogenic enzymes (P450 side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase type 1, and 17α-hydroxylase/17,20 lyase) but produced no effect on steroidogenic acute regulatory protein levels. These results were further confirmed by demonstrating that the knockdown of mTOR using small interfering RNA selectively abrogated the LH/hCG-induced increase in steroidogenic enzyme expression, without affecting steroidogenic acute regulatory protein expression. LH/hCG-stimulated androgen production was also blocked by rapamycin. Furthermore, the pharmacological inhibition of mTORC1 or ribosomal protein S6 kinase 1 signaling prevented the LH/hCG-induced phosphorylation of CREB. Chromatin immunoprecipitation assays revealed the association of CREB with the proximal promoter of the Cyp17a1 gene in response to hCG, and this association was reduced by rapamycin treatment. Taken together, our findings show for the first time that LH/hCG-mediated activation of androgen biosynthesis is regulated by the mTORC1 signaling pathway in T-I cells.

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Complex Role of the Mitochondrial Targeting Signal in the Function of Steroidogenic Acute Regulatory Protein Revealed by Bacterial Artificial Chromosome Transgenesis in Vivo

Molecular Endocrinology ◽

10.1210/me.2007-0493 ◽

2008 ◽

Vol 22 (4) ◽

pp. 951-964 ◽

Cited By ~ 38

Author(s):

Goro Sasaki ◽

Tomohiro Ishii ◽

Pancharatnam Jeyasuria ◽

Youngah Jo ◽

Assaf Bahat ◽

...

Keyword(s):

Bacterial Artificial Chromosome ◽

Adrenal Cortex ◽

Regulatory Protein ◽

Artificial Chromosome ◽

Steroidogenic Acute Regulatory Protein ◽

Mitochondrial Targeting ◽

Targeting Signal ◽

Steroidogenic Cells ◽

Steroidogenic Acute Regulatory

The steroidogenic acute regulatory protein (StAR) stimulates the regulated production of steroid hormones in the adrenal cortex and gonads by facilitating the delivery of cholesterol to the inner mitochondrial membrane. To explore key aspects of StAR function within bona fide steroidogenic cells, we used a transgenic mouse model to explore the function of StAR proteins in vivo. We first validated this transgenic bacterial artificial chromosome reconstitution system by targeting enhanced green fluorescent protein to steroidogenic cells of the adrenal cortex and gonads. Thereafter, we targeted expression of either wild-type StAR (WT-StAR) or a mutated StAR protein lacking the mitochondrial targeting signal (N47-StAR). In the context of mice homozygous for a StAR knockout allele (StAR−/−), all StAR activity derived from the StAR transgenes, allowing us to examine the function of the proteins that they encode. The WT-StAR transgene consistently restored viability and steroidogenic function to StAR−/− mice. Although the N47-StAR protein was reportedly active in transfected COS cells and mitochondrial reconstitution experiments, the N47-StAR transgene rescued viability in only 40% of StAR−/− mice. Analysis of lipid deposits in the primary steroidogenic tissues revealed a hierarchy of StAR function provided by N47-StAR: florid lipid deposits were seen in the adrenal cortex and ovarian theca region, with milder deposits in the Leydig cells. Our results confirm the ability of StAR lacking its mitochondrial targeting signal to perform some essential functions in vivo but also demonstrate important functional defects that differ from in vitro studies obtained in nonsteroidogenic cells.

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Seasonal changes in gonadal expression of gonadotropin receptors, steroidogenic acute regulatory protein and steroidogenic enzymes in the European sea bass

General and Comparative Endocrinology ◽

10.1016/j.ygcen.2009.03.023 ◽

2009 ◽

Vol 162 (3) ◽

pp. 265-275 ◽

Cited By ~ 92

Author(s):

Ana Rocha ◽

Silvia Zanuy ◽

Manuel Carrillo ◽

Ana Gómez

Keyword(s):

Seasonal Changes ◽

Regulatory Protein ◽

Sea Bass ◽

Steroidogenic Acute Regulatory Protein ◽

Steroidogenic Enzymes ◽

European Sea Bass ◽

Gonadotropin Receptors ◽

Steroidogenic Acute Regulatory

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StarD7 Gene Expression in Trophoblast Cells: Contribution of SF-1 and Wnt-β-Catenin Signaling

Molecular Endocrinology ◽

10.1210/me.2010-0503 ◽

2011 ◽

Vol 25 (8) ◽

pp. 1364-1375 ◽

Cited By ~ 9

Author(s):

Viviana Rena ◽

Jésica Flores-Martín ◽

Sofía Angeletti ◽

Graciela M. Panzetta-Dutari ◽

Susana Genti-Raimondi

Keyword(s):

Gene Expression ◽

Intracellular Transport ◽

Regulatory Protein ◽

Steroidogenic Acute Regulatory Protein ◽

Regulatory Sequences ◽

Physical Interaction ◽

Lipid Transfer ◽

Amino Acid Mutations ◽

Steroidogenic Acute Regulatory

Steroidogenic acute regulatory protein-related lipid transfer domain containing 7 (StarD7) is a poorly characterized member of the steroidogenic acute regulatory protein-related lipid transfer proteins, up-regulated in JEG-3 cells, involved in intracellular transport and metabolism of lipids. Previous studies dealing with the mechanisms underlying the human StarD7 gene expression led us to define the cis-acting regulatory sequences in the StarD7 promoter using as a model JEG-3 cells. These include a functional T cell-specific transcription factor 4 (TCF4) site involved in Wnt-β-catenin signaling. To understand these mechanisms in more depth, we examined the steroidogenic factor 1 (SF-1) contribution to StarD7 expression. Cotransfection experiments in JEG-3 cells point out that the StarD7 promoter is activated by SF-1, and this effect is increased by forskolin. EMSA using JEG-3 nuclear proteins demonstrated that SF-1 binds to the StarD7 promoter. Additionally, chromatin immunoprecipitation analysis indicated that SF-1 and β-catenin are bound in vivo to the StarD7 promoter. Reporter gene assays in combination with mutations in the SF-1 and TCF4 binding sites revealed that the StarD7 promoter is synergistically activated by SF-1 and β-catenin and that the TCF4 binding site (−614/−608) plays an important role in this activation. SF-1 amino acid mutations involved in the physical interaction with β-catenin abolished this activation; thus demonstrating that the contact between the two proteins is necessary for an efficient StarD7 transcriptional induction. Finally, these data suggest that β-catenin could function as a bridge between SF-1 and TCF4 forming a ternary complex, which would stimulate StarD7 expression. The SF-1 and β-catenin pathway convergence on StarD7 expression may have important implications in the phospholipid uptake and transport, contributing to the normal trophoblast development.

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