Regulation of LH receptor mRNA binding protein by miR-122 in rat ovaries

Abstract

LH receptor (LHR) expression in the ovary is regulated by the RNA binding protein, (LHR mRNA binding protein [LRBP]), which has been identified as being mevalonate kinase. This study examined the role of microRNA miR-122 in LRBP-mediated LHR mRNA expression. Real-time PCR analysis of ovaries from pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-primed female rats treated with hCG to down-regulate LHR expression showed that an increase in miR-122 expression preceded LHR mRNA down-regulation. The expression of miR-122 and its regulation was confirmed using fluorescent in situ hybridization of the frozen ovary sections using 5'-fluorescein isothiocyanate-labeled miR-122 locked nucleic acid probe. The increased expression of miR-122 preceded increased expression of LRBP mRNA and protein, and these increases were followed by LHR mRNA down-regulation. Inhibition of protein kinase A (PKA) and ERK1/2 signaling pathways by H89 and UO126, respectively, attenuated the hCG-mediated up-regulation of miR-122 levels. This was also confirmed in vitro using human granulosa cells. These results suggest the possibility that hCG-mediated miR-122 expression is mediated by the activation of cAMP/PKA/ERK signaling pathways. Inhibition of miR-122 by injection of the locked nucleic acid-conjugated antagomir of miR-122 abrogated the hCG-mediated increases in LRBP protein expression. Because it has been previously shown that miR-122 regulates sterol regulatory element-binding proteins (SREBPs) and SREBPs, in turn, regulate LRBP expression, the role of SREBPs in miR-122-mediated increase in LRBP expression was then examined. The levels of active forms of both SREBP-1a and SREBP-2 were increased in response to hCG treatment, and the stimulatory effect was sustained up to 4 hours. Taken together, our results suggest that hCG-induced down-regulation of LHR mRNA expression is mediated by activation of cAMP/PKA/ERK pathways to increase miR-122 expression, which then increases LRBP expression through the activation of SREBPs.

Figure 1.

Analysis of miR-122 expression in the ovaries using real-time PCR and fluorescent in situ hybridization analysis. A, Superovulated rats were injected with hCG on day 5. Ovaries were collected 0 and 30 minutes and 1, 2, 3, and 4 hours later. Total RNA was isolated from the ovaries and the levels of miR-122 and U6 snRNA were determined by real-time PCR using predesigned primers and probes as described in Materials and Methods. The graphs represent changes in miR-122 levels normalized to U6 small nuclear RNA and shown as percent change vs CTL. Error bars represent mean ±SE. *, P < .05, n = 3. B, Fluorescent in situ hybridization analysis of CTL and hCG-treated (1 hour) frozen rat ovary sections were conducted using FITC-labeled miR-122 LNA probe as described in detail in Materials and Methods. Each panel shows 4′,6-diamidino-2-phenylindole staining (A, A*, C & C*; blue fluorescence) on the left and FITC (miR-122) staining (B, B*, D & D*, green fluorescence) on the right. Top and bottom panels show ×60 and ×100 magnifications, respectively. Experiment was repeated 4 times with same results.

Figure 2.

Temporal relationship of miR-122, LRBP, and LHR mRNA expressions in the ovary following hCG treatment. Superovulated rats were injected with hCG on day 5 and ovaries were collected 0 and 30 minutes and 1, 2, 3, 4, 6, and 12 hours later. Equal amounts of RNA from the CTL or hCG-treated ovaries were subjected to real-time PCR using predesigned primers and probes for rat LRBP or LHR mRNA (A and C) as described in Materials and Methods. The graphs represent ratio of LRBP or LHR mRNA levels normalized to 18S RNA and are shown as percent change vs CTL. Error bars represent mean ± SE. *, P < .05; n = 4. Equal amounts of protein from the CTL or hCG-treated S10 fractions were subjected to Western blot analysis using LRBP antibody (B). The membranes were stripped and reprobed for tubulin (bottom lane). The blots shown are representative of 3 independent experiments. The graphs represent LRBP levels normalized to tubulin and are shown as percent change vs control. Error bars represent mean ± SE. *, P < .05; n = 4. The graphic representation of the relationship between the time course of miR-122, LRBP, and LHR mRNA expressions after hCG treatment is shown in panel D.

Figure 3.

Inhibition of PKA and ERK1/2 inhibits hCG-induced increases in miR-122 expression in the rat ovaries. Superovulated rats were injected with H-89 (100 mg/kg body weight) or UO126 (10 mg/kg body weight) 1 hour before hCG injection on day 5 and ovaries were collected 1 hour later. Total RNA was isolated from the ovaries, and the levels of miR-122 and U6 small nuclear RNA (CTL) were determined by real-time PCR using predesigned primers and probes as described in Materials and Methods. The graphs represent changes in miR-122 levels normalized to U6 small nuclear RNA and shown as percent change vs CTL. Error bars represent mean ± SE. *, P < .05; n = 3.

Figure 4.

Inhibition of PKA and ERK1/2 inhibits hCG-induced increases in miR-122 expression in human granulosa cells. Day 4 granulosa cells were serum starved, treated with hCG (10 IU/mL) with or without the ERK1/2 inhibitor UO126 (10 μM; 1-hour pretreatment) for a total of 1 hour and were processed for RNA isolation as described in Materials and Methods. RNAs were reverse transcribed, and the resulting cDNAs were subjected to real-time PCR quantitation using specific primers and probes for miR-122 and U6 small nuclear RNA. The graphs represent changes in miR-122 levels normalized to U6 snRNA and shown as percent change vs CTL. Error bars represent mean ± SE. *, P < .05 vs CTL; ^#, P < .05 vs hCG; n = 4.

Figure 5.

Inhibition of miR-122 abrogated hCG-induced increases in LRBP expression. LNA-conjugated miR-122 antagomir was injected into the bursa of the ovaries of superovulated rats on day 4 followed by hCG on day 5. Ovaries were collected 6 hours later. Equal amounts of protein from the CTL, hCG-treated, or antagomir + hCG-treated ovaries were subjected to real-time PCR using predesigned primers and probes for miR-122 (upper panel) as described in Materials and Methods. The graphs represent ratio of miR-122 levels normalized to U6 small nuclear RNA and are shown as percent change vs CTL. Error bars represent mean ± SE. Equal amounts of protein from the CTL, hCG-treated, or antagomir + hCG-treated S10 fractions were subjected to Western blot analysis using LRBP antibody (lower panel, top lane). The membranes were stripped and reprobed for tubulin (lower panel, bottom lane).

Figure 6.

Activation of SREBP-1a and SREBP-2 during hCG-induced LHR mRNA down-regulation. Superovulated rats were injected with hCG on day 5, and ovaries were collected 1, 2, 3, 4, and 5 hours later. S10 fractions were prepared using radioimmune immunoprecipitation assay buffer. Equal amounts of protein from the CTL or hCG-treated S10 fractions were subjected to Western blot analysis using SREBP-2 antibody. The blots were stripped and reprobed first with SREBP-1a and then with tubulin. The blots shown are representative of 3 independent experiments. The graphs represent SREBP levels normalized to tubulin and are shown as percent change vs control. Error bars represent mean ± SE. *, P < .05; n = 3.

Figure 7.

Schematic model depicting the proposed signaling pathway in LH/hCG-induced miR-122-mediated LHR mRNA down-regulation. Binding of ligand to LHR induces activation of ERK1/2 through the cAMP/PKA pathway. This leads to an increase in the expression of miR-122, which causes the activation of SREBPs through its interaction with unidentified targets. This is followed by the increased expression of LRBP and its LHR mRNA binding activity, which ultimately results in LHR mRNA degradation. Direct activation is shown by solid arrows and indirect interaction/activation is shown by broken arrows.