Date Published: October 28, 2013
Publisher: Public Library of Science
Author(s): Zhigang Hu, Wen-Jun Shen, Yuan Cortez, Xudong Tang, Li-Fen Liu, Fredric B. Kraemer, Salman Azhar, Bernard Mari.
Given the emerging roles of miRNAs as potential posttranscriptional/posttranslational regulators of the steroidogenic process in adrenocortical and gonadal cells, we sought to determine miRNA profiles in rat adrenals from animals treated with vehicle, ACTH, 17α-E2 or dexamethasone. Key observations were also confirmed using hormone (Bt2cAMP)-treated mouse Leydig tumor cells, MLTC-1, and primary rat ovarian granulosa cells.
RNA was extracted from rat adrenal glands and miRNA profiles were established using microarray and confirmed with qRT-PCR. The expression of some of the hormone-sensitive miRNAs was quantified in MLTC-1 and granulosa cells after stimulation with Bt2cAMP. Targets of hormonally altered miRNAs were explored by qRT-PCR and Western blotting in adrenals and granulosa cells.
Adrenals from ACTH, 17α-E2 and dexamethasone treated rats exhibited miRNA profiles distinct from control animals. ACTH up-regulated the expression of miRNA-212, miRNA-182, miRNA-183, miRNA-132, and miRNA-96 and down-regulated the levels of miRNA-466b, miRNA-214, miRNA-503, and miRNA-27a. The levels of miR-212, miRNA-183, miRNA-182, miRNA-132, miRNA-370, miRNA-377, and miRNA-96 were up-regulated, whereas miR-125b, miRNA-200b, miR-122, miRNA-466b, miR-138, miRNA-214, miRNA-503 and miRNA27a were down-regulated in response to 17α-E2 treatment. Dexamethasone treatment decreased miRNA-200b, miR-122, miR-19a, miRNA-466b and miRNA27a levels, but increased miRNA-183 levels. Several adrenal miRNAs are subject to regulation by more than one hormone. Significant cAMP-induced changes in certain miRNAs were also noted in MLTC-1 and granulosa cells. Some of the hormone-induced miRNAs in steroidogenic cells were predicted to target proteins involved in lipid metabolism/steroidogenesis. We also obtained evidence that miR-132 and miRNA-214 inhibit the expression of SREBP-1c and LDLR, respectively.
Our results demonstrate that expression of a number of miRNAs in steroidogenic cells of the testis, ovary and adrenal glands is subject to hormonal regulation and that miRNAs and their regulation by specific hormones are likely to play a key role in posttranscriptional/posttranslational regulation of steroidogenesis.
Steroid hormones, which are synthesized most prominently in the adrenal gland and gonads –, play important roles in the regulation of carbohydrate, lipid and protein metabolism and immune function (glucocorticoids), salt and water balance and blood pressure regulation (mineralocorticoids) and maintenance of secondary sex characteristics, reproductive functions and muscle and bone growth (testosterone, progestins and estrogens) . Steroidogenesis or biosynthesis of steroid hormones represents a complex multistep and multienzymes process by which precursor cholesterol is converted to pregnenolone and subsequently metabolized into other biologically active steroids in a tissue specific manner –. This process can be broadly divided into five major steps: 1) acquisition of cholesterol from exogenous (lipoproteins) and endogenous (de novo synthesis) sources for storage in the form of cholesterol esters (CEs) in lipid droplets, 2) mobilization of cholesterol from lipid droplet stored CEs, 3) transport of cholesterol to and from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM), where cytochrome P450 side chain cleavage enzyme (P450scc, encoded by CYP11A1) is localized, 4) P450scc catalyzed cleavage of a 6-carbon unit from the cholesterol side chain producing pregnenolone, the common precursor – for the synthesis of all of the other steroid hormones, and 5) efflux of pregnenolone from the mitochondria to the endoplasmic reticulum (ER), where it is converted by ER enzymes into intermediate precursors, which further shuttle between mitochondria and ER for the tissue specific production of progestins, estrogens, androgens, glucocorticoids or mineralocorticoids , .
Steroid hormone synthesis occurs predominantly in the steroidogenic cells of the adrenal gland, ovary and testis and is under the control of trophic peptide hormones secreted from the pituitary. The rate limiting step in steroidogenesis is the trophic hormone−/cAMP-stimulated and StAR-mediated translocation of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane where the side-chain cleavage enzyme (P450scc; Cyp11A1) carries out the first committed step in steroidogenesis, i.e., conversion of cholesterol to pregnenolone –. This step is subject to both acute , – and chronic , , – stimulation, and trophic hormones regulate this step mainly at the level of gene transcription. Although limited information is also available to suggest that posttranscriptional and posttranslational events may be involved in the regulation of steroidogenesis, relatively little information is available on the biological factors that possibly mediate these events. Emerging evidence showing hormonal regulation of miRNAs in steroidogenic cells , –, coupled with the identification of a diverse and large number of miRNAs –, strongly suggest that miRNAs may be involved in the posttranscriptional/posttranslational regulation of steroidogenesis. In this study, we first carried out a comprehensive analysis of miRNA profiling using control and in vivo hormone treated rat adrenals to identify miRNAs whose expression is altered in response to ACTH, 17α-ethinyl estradiol (17α-E2) or dexamethosone (DEX) treatment. Taking cues from the adrenal data, we also examined the effects of Bt2cAMP (the second messenger of trophic hormone action) stimulation of rat ovarian granulosa cells and mouse testicular Leydig tumor cells, MLTC-1, on the expression of some of the relevant miRNAs.