miR-449a Contributes to Glucocorticoid-Induced CRF-R1 Downregulation in the Pituitary During Stress

Abstract

The hypothalamic-pituitary-adrenal axis is controlled by the feedback of glucocorticoids on the hypothalamus and pituitary. Stress increases CRF, ACTH, and glucocorticoid secretion. The expression of not only CRF mRNA in the hypothalamus and proopiomelanocortin mRNA in corticotrophs, but also CRF type 1 receptor (CRF-R1) mRNA and protein on corticotrophs are downregulated through glucocorticoids. However, the mechanisms underlying the glucocorticoid-induced CRF-R1 downregulation are not fully understood. Short RNA molecules, called microRNAs (miRNAs), are posttranscriptional regulators that usually induce translational repression or gene silencing via binding to complementary sequences within target mRNAs. We hypothesized that glucocorticoids may induce the expression of miRNAs in the pituitary, which are involved in glucocorticoid-induced downregulation of CRF-R1. We found 3 miRNAs with sequences predicted to bind to the CRF-R1 3' untranslated region (3'-UTR) by database search. Expression of 1 of these miRNAs (miR-449a) was significantly higher in the anterior pituitary of restrained rats than in that of unrestrained control rats. Expression of miR-449a was evident in many anterior pituitary cells, including corticotrophs. Although overexpression of miR-449a decreased CRF-R1 mRNA and CRF-R1 protein expression, knockdown of miR-449a attenuated dexamethasone-induced suppression of CRF-R1 mRNA and CRF-R1 protein expression in the monolayer-cultured pituitary cells. Notably, luciferase activity was significantly lower in cells cotransfected with a luciferase vector containing the CRF-R1 3'-UTR and a miR-449a vector. miR-449a expression was significantly increased by dexamethasone. Adrenalectomy attenuated restraint-induced increase in miR-449a expression in the pituitary. These results indicated that miR-449a plays an important role in stress-induced, glucocorticoid-mediated downregulation of CRF-R1 expression.

Impaired miR449a-induced downregulation of Crhr1 expression in low-birth-weight rats

“…The expression of miR449a in the anterior pituitary was increased by restraint in NBW offspring but not in LBW offspring. We have previously demonstrated that overexpression of miR449a suppressed mRNA and protein expression of Crhr1, and knockdown of miR449a blocked dexamethasone-induced suppression of Crhr1 expression (Nemoto et al 2013), indicating that miR449a suppresses Crhr1 expression. Results of a previous in vitro study had also indicated that the expression of miR449a was positively regulated by glucocorticoids, but not by CRF.…”

“…SDS sample buffer (200 mM Tris-HCl, pH 6.8, 30% glycerol, 6% SDS, 0.03% bromophenol blue, and 3% b-mercaptoethanol) and boiled for 5 min. They were then separated by SDS-PAGE and transferred onto (Nemoto et al 2013). Membranes were washed and incubated with HRP-conjugated antigoat IgG (Jackson Immuno Research Laboratory, West Grove, PA, USA).…”

“…We detected Crhr1 protein expression using anti-CRF receptor antibodies as described previously (Nemoto et al 2013; Fig. 1E, F, and G).…”

“…We then examined the expression of miR449a, which we have previously demonstrated to be involved in Crhr1 downregulation (Nemoto et al 2013), in the anterior pituitary of restrained LBW or normal-birth-weight (NBW) rats. As microRNAs (miRNAs) have been recently shown to be present in exosomes released from cells and those exosomal miRNAs are stable in the blood or extracellular fluids (Hunter et al 2008, Turchinovich et al 2011, we attempted to detect exosomal miR449a in blood and culture medium of rat anterior pituitary cells.…”

A mikro-RNS-ek szerepe az agyalapimirigy-daganatok tumorbiológiájában

“…Egy másik tanulmány-ban az egér központi idegrendszerének különböző részeit vizsgálva sikerült agyalapimirigyre specifikus miRNS-eket azonosítani [22]. Amellett, hogy a miRNSeknek az emberben is fontos szerepük van a normál mirigy fejlődésében [19,23,24] és hormontermelésében [25][26][27][28][29][30][31], az elmúlt években egyre több humán vizsgálat támasztja alá azt is, hogy részt vehetnek az adenomák kialakulásában és növekedésében [32][33][34][35].…”