Eukaryotic Initiation Factor 5A Plays an Essential Role ...

ORIGINAL

RESEARCH

Eukaryotic Initiation Factor 5A Plays an Essential Role in Luteinizing Hormone Receptor Regulation Bindu Menon1, Thippeswamy Gulappa1, and K. M. J. Menon* Departments of Obstetrics/Gynecology and Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI - 48109 – 0617

Downregulation of LH receptor (LHR) in the ovary by its ligand is mediated by a specific RNA binding protein designated as LHR mRNA binding protein (LRBP), through translational suppression and mRNA degradation. Using yeast two hybrid screens, we previously identified eukaryotic initiation factor 5A (eIF5A) as one of the proteins that interacts with LRBP during LHR mRNA downregulation. The present study examined the role of eIF5A and its hypusination in the context of LHR mRNA downregulation. The association of eIF5A with LRBP or LHR mRNA was determined using immunoprecipitation and RNA Immunoprecipitation (RNA-IP) assays. The results showed that the association of eIF5A with LHR mRNA-LRBP complex increased significantly during downregulation. Furthermore, gel fractionation and hypusination activity assay both showed increased hypusination of eIF5A during LHR mRNA downregulation. Abolishment of hypusination by pretreatment with the chemical inhibitor, GC7 prevented the association of eIF5A with LHR mRNA and LRBP. Inhibition of hypusination also reduced the extent of ligand-induced downregulation of LHR mRNA as well as the expression of functional LH receptors assessed by real time PCR and 125 I-hCG binding assays, respectively. Loss of hCG-mediated downstream signaling during LH receptor downregulation was also restored by inhibition of hypusination of eIF5A. Thus, the present study, for the first time reveals the crucial role of eIF5A and its hypusination in the regulation of LH receptor expression in the ovary.

uteinizing hormone receptor (LHR) plays an essential role in reproduction (1– 4). In the ovary, LHR expression shows dynamic changes during the ovarian cycle. For instance, its levels increase progressively during follicular phase but decrease dramatically following preovulatory surge and a full recovery reaching maximal levels at midluteal phase (4 –7). The downregulation of the receptor can also be mimicked by the administration of a pharmacological dose of its ligands, LH or hCG to the superovulated rats. Past studies from our laboratory have shown that the downregulation of the receptor occurs through a post-transcriptional mechanism involving the association of LHR mRNA with an RNA binding protein, designated as LHR mRNA binding protein (LRBP) (8) which was later characterized as being mevalonate kinase (9, 10). The expression of LHR mRNA and LRBP exhibit a reciprocal relationship in the ovaries during up and down-

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regulation of LHR (10). Further studies showed that during downregulation, LRBP binds to the coding region of LHR mRNA to impede translation, resulting in translocation of the ribonucleoprotein complex to Processingbodies (P-bodies), where it undergoes decapping and degradation (11–13). In order to understand the possible involvement of other components in LHR downregulation, we performed yeast two hybrid assays to determine if LRBP interacts with other proteins that lead to LHR mRNA degradation. These studies identified Eukaryotic Initiation Factor 5A (eIF5A) as an interacting partner with LRBP, providing potential mechanistic insight into LHR downregulation (14). However, the role of eIF5A in LHR mRNA downregulation is not understood. Several studies have shown that eIF5A participates in many aspects of RNA metabolism including mRNA decay, transport and most recently, translation elongation (15–22).

ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received April 24, 2014. Accepted September 8, 2014.

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doi: 10.1210/me.2014-1132

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Role of eIF5A in LHR mRNA regulation

Furthermore, eIF5A has been known to undergo hypusination, a unique post-translational modification, which is essential for many of its functions (23, 24) including RNA binding (25). The goal of the present study was to examine the role of eIF5A and its hypusination in LH receptor downregulation in the ovary. Here we provide evidence that eIF5A plays an essential role in ligand-mediated downregulation of LHR mRNA. Furthermore, inhibition of eIF5A hypusination showed a significant reduction in the extent of ligand-mediated LHR mRNA downregulation as well as cell surface receptor expression and downstream signaling.

Materials and Methods Materials. Pregnant mare serum gonadotropin (PMSG) was obtained from Calbiochem (San Diego, CA). Highly purified human chorionic gonadotropin (hCG; CR 127) was purchased from Dr. A. F. Parlow through National Hormone and Peptide Program (Torrance, CA). EDTA-free protease inhibitor mixture tablets and RNAse inhibitor (rRNasin) were the products of Roche Applied Science (Indianapolis, IN) and Promega (Madison, WI) respectively. Real time PCR primers for LH receptor and 18S rRNA (TaqMan Assay-on-Demand Gene Expression Product) as well as Multiscribe reverse transcriptase were obtained from Applied Biosystems (Foster City, CA). Since LRBP was identified as MVK, anti-N-terminal mevalonate kinase IgG was raised against the first 15 N-terminal amino acids of MVK (MLSEVLLVSAPGKVI) and this antibody is referred to as the LRBP antibody in the text. Purified antibodies against ␤ tubulin and eIF5A (catalog number E1783) were commercial products (Sigma, St. Louis, MO). Recombinant eIF5A was purchased from Prospec Bio (Rehovot, Israel) and [3H]-spermidine from Perkin Elmer (Waltham, MA). The Super Signal West Femto chemiluminescence kit and antirabbit/anti mouse IgG conjugated to horseradish peroxidase were obtained from Pierce (Rockford, IL). BCA reagent was purchased from GE Healthcare Life Sciences (Piscataway, NJ). Animals and Tissues. Superovulation was induced in 23 day old female Sprague-Dawley rats by subcutaneous injection of 50 IU of PMSG followed by 25 IU of hCG 56 hours later. The day of hCG injection was taken as day 0. LH receptor downregulation was induced by the injection of 50 IU of hCG on day 5. Ovaries were collected 0, 2, 4, 6, 8 and 12h after hCG injection and were frozen in liquid nitrogen until further use. To inhibit eIF5A hypusination, rats were treated i.p. with GC7 (16 mg/kg b.w.) 2 hours prior to hCG administration, based on preliminary experiments and previously published work (26). Preparation of Polysomes. Polysomes were isolated using a previously published procedure (13). Briefly, ovaries from control and hCG-injected (with or withour GC7 pretreatment) rats were homogenized in solution A (1 mM potassium acetate, 2 mM Mg(Ac)2, 2 mM dithiothreitol, and 10 mM Tris acetate, pH 7.6) at 4°C. After centrifugation at 10,000 ⫻ g for 10 minutes, the supernatants were layered over a cushion of solution B (so-

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lution A containing 30% sucrose) and centrifuged at 130,000 ⫻ g for 2.5 hours. The polyribosome pellets were resuspended in solution A and stored at 80°C until use. Immunoprecipitation (IP). Ovaries were homogenized in RIPA buffer and the homogenates were centrifuged at 10,000 x g for 10 minutes to obtain cytosolic S10 fractions. Equal amounts of protein from each sample (S10 fractions or polysomes) were incubated overnight in primary antibody against the protein of interest at 4°C followed by incubation with Protein A- Agarose beads for two hours at 4°C. The immunoprecipitated proteins were then subjected to SDS-PAGE and Western Blot analysis. To examine whether LHR mRNA is necessary for the recognition of LRBP by eIF5A, polysomes were incubated with RNase A (50 ␮g/ml final concentration) for 10 minutes at 37°C before incubation with the primary antibody. Western Blot analysis. For this, tissue homogenates (S10 fractions) in RIPA buffer or immunoprecipitates were subjected to 10% SDS-PAGE under reducing conditions followed by Western blot analysis as previously described (10). The presence of immune complexes was detected by following chemiluminescence signals. RNA Immunoprecipitation (RIP). Ovaries were homogenized in NET-2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Nonidet P-40) containing RNasin (100 U/0.5 ml of buffer) and protease inhibitors. The homogenates were centrifuged at 10,000 x g for 10 minutes at 4°C. The supernatants were collected and 1–1.5 mg of protein/ml from the extracts (S10 fractions) was used for immunoprecipitation of RNP complex as described previously (12, 13). Real-Time PCR (qPCR) analysis. Total RNAs were reversetranscribed and subjected to real time PCR quantitation as described before (13, 27). Briefly, aliquots (50 –100 ng) of total RNA (extracted from ovaries or from immunoprecipitates) were reverse transcribed using 2.5 ␮M random hexamer, 500 ␮M deoxynucleotide triphosphates, 5.5 mM MgCl2, 8 U ribonuclease inhibitor, and 25 U Multiscribe reverse transcriptase. The resulting cDNAs were diluted with water. The real-time PCR quantitation was then performed using 5 ␮l of the diluted cDNAs in triplicate using predesigned primers and probes. Reactions were carried out in a volume of 25 ␮l using Applied Biosystems 7300 Real-Time PCR system for 40 cycles (95°C for 15 seconds, 60°C for 1 minute) after initial incubation for 10 minutes at 95°C. The fold change in gene expression was calculated using the standard curve method with 18S rRNA as the internal control using the ⌬⌬Ct method (28). Hypusine-formation assay. In vitro hypusination activity assay was performed using previously published procedure (26, 29). Briefly, ovarian S10 fractions (40 ␮g protein) were added to assay mixture containing 50 mM glycine, pH 8.3, 20% glycerol, 2 mM DTT, 150 mM KCl, 10 mM MgCl2, 0.1 mg/ml BSA, 0.1 mM NAD⫹, 20 ng of recombinant eIF5A protein and 2.0 ␮Ci of [3H]-spermidine in a final volume of 25 ␮l. The reaction mixture was incubated at 37°C for 2h. The reaction was terminated by the addition of 5␮l of SDS-PAGE sample buffer. The proteins were separated on a 12% SDS-PAGE gel, transferred to nitro-


doi: 10.1210/me.2014-1132

cellulose membrane and subjected to flurography using Kodak BioMax TranScreen LE and BioMax MS film at –70°c for 72 hours. The images were then scanned and quantitated.


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1B). As expected, the levels of total LHR mRNA started to decline at 4h of hCG treatment declining further to

Preparation of membrane fractions. A partially purified membrane fraction was prepared as described previously from our laboratory (30). Briefly, ovaries were homogenized in 5 vol of PBS (0.15 M NaCl-0.05 M phosphate pH 7.4) and centrifuged at 400 g for 4 minutes. The pellet was washed once, and the combined supernatants were centrifuged at 700 g for 10 minutes. The pellet was again washed once, and the combined supernatants were centrifuged at 10000 g for 30 minutes. The resulting pellet was suspended in 4 vol PBS and stored at –70°c until used. 125 I-hCG binding assay. Radioiodination of hCG was performed using chloramine T method to give a specific activity of 40 – 60 cpm/pg as described previously (31). Binding of 125IhCG to the membrane fractions isolated from rat ovaries was performed as described previously (30, 31) by incubating at 37°C for 90 minutes with increasing concentrations of 125I-hCG (10 –240 ng). Nonspecific binding was determined by performing binding assays in the presence of 1000 fold excess of unlabeled hCG. Maximal binding capacity (Bmax) and equilibrium dissociation constant (Kd) were calculated from the binding data using Scatchard analysis as described previously from our laboratory (31).

Statistical Analysis. Statistical analysis was carried out using one-way ANOVA followed by the Tukey multiple comparison test. Each experiment was repeated at least three times with similar results. Blots and autoradiograms shown are representative of a minimum of three experiments.

Results Association of eIF5A with LRBP and LHR mRNA during hCG-induced LHR mRNA downregulation. We recently identified eIF5A as an LRBP interacting protein in a yeast two-hybrid screen (14). To examine the dynamics of eIF5A interaction with LRBP and LHR mRNA in ovaries, we performed immunoprecipitation and RNA-immunoprecipitation (RNA-IP) of S10 fractions from control and LHR downregulated rat ovaries using eIF5A antibody. The immunoprecipitates were then subjected to Western Blot using LRBP antibody and total RNA isolated from RNA-IP were subjected to real time PCR using rat LHR specific primers. The results presented in Figure 1A show that there was a significant increase in the formation of eIF5A-LRBP complex following 4 and 6h of hCG treatment to downregulate LHR. The levels of eIF5A-LRBP complex was 2.7-fold higher in the hCG treated samples compared to control after 4h of hCG treatment, which showed a further increase at 6h to produce a 4-fold difference vs control. The association of LHR mRNA with eIF5A also showed an increase during this period (Figure

FIGURE 1. eIF5A associates with LHR mRNA and LRBP during LHR downregulation. A, 23 day old female rats were treated s.c. with hCG on the fifth day of superovulation, ovaries were collected 0, 4 and 6 hours later and cytosolic extracts (S10) prepared. (A) Equal amounts of protein from the control (CTL) or hCG-treated S10 fractions were subjected to immunoprecipitation using eIF5A antibody. The immunoprecipitates were subjected to Western Blot analysis using LRBP antibody (upper panel). The membranes were stripped and reprobed for eIF5A (lower panel). The input was also analyzed for eIF5A, LRBP and ␤-tubulin (panels 3, 4 and 5). The blots shown are representative of four independent experiments. The graphs represent LRBP levels normalized to eIF5A and are represented as fold change vs. control. Error bars represent mean ⫾SE. *P ⬍ .05. (B) Equal amounts of protein from the control (CTL) or hCG-treated S10 fractions were subjected to RNA immunoprecipitation using antibodies against eIF5A followed by real time PCR using predesigned primers and probes for rat LH receptor mRNA as described in Methods. Total LHR mRNA present in the S10 fractions were also measured using real time PCR. The graphs represent ratio of LHR mRNA levels in the immunoprecipitate normalized to total LHR mRNA and are presented as fold change vs. control. Error bars represent mean ⫾SE. *P ⬍ .05, n ⫽ 3.


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insignificant levels by 24h confirming that downregulation of LHR mRNA occurred during this time period (data not shown). Consistent with this, the association of LHR mRNA with eIF5A showed a significant increase at 4 and 6h following hCG treatment. Since the extent of downregulation was minimum at 4 and 6 hours as has been shown previously by us (32–34), we used these time periods to determine the early events that lead to LHR mRNA degradation. There was a 5-fold increase in the levels of eIF5A-LHR mRNA complex at 4h, which further increased to 7.2 fold by 6h of hCG treatment (Figure 1B). Together, these results showed that the association of eIF5A with the LHR mRNA-LRBP complex increases significantly during LHR downregulation. Association of eIF5A with LRBP and LHR mRNA in isolated ribosomes during LHR downregulation. We have previously shown that LRBP binds to LHR mRNA in the ribosomes to cause translational suppression of LHR mRNA (12, 13). Since eIF5A has also been shown to physically interact with protein components of the translational machinery in the ribosomes (35), studies were conducted to determine the association of eIF5A-LRBPLHR mRNA complex in the ribosomes. Ribosomes were isolated as described previously (13) from control and downregulated (4 and 6h) ovaries and immunoprecipitated with eIF5A antibody and the immunoprecipitates were then subjected to real time PCR (Figure 2A) or Western Blot analysis (Figure 2B). The purity of ribosomes were confirmed by the presence of ribosomal protein S6 and absence of the cytosolic protein, heat shock protein 90␤, assessed by Western Blot analysis (data not shown) (13). RNA immunoprecipitation followed by real time PCR analysis showed that the amount of LHR mRNA bound to eIF5A in the ribosomes was higher in the downregulated samples compared to control (Figure 2A). The levels of LHR mRNA-eIF5A complex was 3-fold higher in the 4h hCG treated samples compared to control and increased further to reach an 8-fold vs. control by 6h of hCG treatment. Western Blot analysis of the immunoprecipitates using LRBP antibody showed that there was increased association of LRBP with eIF5A in the ribosomes during the initiation of LHR downregulation (Figure 2B, panel 1). There was a 2.8-fold increase at 4h and 5- fold increase at 6h (Panel 1) following hCG treatment (P ⬍ .05 vs. control). These results show that eIF5A specifically interacts with LHR mRNA-LRBP complex in the ribosomes and the extent of this association increased with progress in downregulation. In order to determine if eIF5A associates with LRBP independent of LHR mRNA, polysomes were treated with RNAse A prior to immunoprecipitation to remove LHR mRNA. The results showed

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that the association of eIF5A with LRBP was not affected by RNAse treatment. The binding of LRBP and eIF5A at 4h and 6h of hCG treatment was higher than the untreated controls (Figure 2B, panel 3), suggesting that prior association with LHR mRNA does not appear to be nec-

FIGURE 2. Association of eIF5A with LHR mRNA and LRBP in the polysomes. 23 day old female rats were treated with a single dose of hCG on the fifth day of superovulation, ovaries were collected 0, 4 and 6 hours later and polysomes were isolated as described in Methods. (A) Equal amounts of protein from the control (CTL) or hCG-treated polysomes were subjected to immunoprecipitation using eIF5A antibody followed by real time PCR using predesigned primers and probes for rat LH receptor mRNA as described in Methods. Total LHR mRNA levels present in the polysomes fractions were also measured using real time PCR. The graphs represent ratio of LHR mRNA levels in the immunoprecipitate to total LHR mRNA and are presented as fold change vs. control. Error bars represent mean ⫾SE. *P ⬍ .05, n ⫽ 4. (B) Equal amounts of protein from the control (CTL) or hCG-treated polysomes, with (Panels 3 and 4) or without RNAse treatment (panels 1 and 2), were subjected to immunoprecipitation using eIF5A antibody followed by Western Blot analysis of the immunoprecipitates using LRBP antibody (upper panel). The membranes were stripped and reprobed for eIF5A (lower panel). The input was also analyzed for eIF5A and LRBP (panels 5 and 6). The blots shown are representative of four independent experiments. The graphs represent LRBP levels normalized to eIF5A and are presented as fold change vs. control. Error bars represent mean ⫾SE. *P ⬍ .05.


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essary for the interaction of LRBP with eIF5A. This result is relevant in light of our previous findings that the association of LRBP with LHR mRNA causes translational suppression of LHR (12). Expression of hypusinated eIF5A is increased during LHR downregulation. eIF5A has been shown to undergo hypusination, a unique post-translational modification by transferring 4-amino butyl moiety from spermidine to ⑀-amino group of lysine of eIF5A to convert eIF5A to its active form (36). The reaction occurs in two steps. First, deoxyhypusine synthase adds 4-amino butyl group from spermidine to the lysine residue 50 of eIF5A and then hydroxylation of 4-amino-butyl group by deoxyhypusine hydroxylase (36). We first sought to examine if the levels of hypusinated eIF5A is increased by hCG during LHR mRNA downregulation. Ovaries from superovulated rats treated with hCG were harvested and extracts were analyzed using Western Blot. Unhypusinated (pre) eIF5A (molecular weight 17 kDa) migrates faster in the gel and appears as a band directly below the hypusinated (hyp) form in Western blot analysis (Figure 3A) (37). Densitometric analysis of the blot showed that there was a 100% percent increase in the intensity of the band representing hypusinated eIF5A at both 4 and 6h of downregulation. Since hypusination of eIF5A has been shown to be inhibited by GC7, a cell-permeable inhibitor that targets the spermidine-binding site of deoxyhypusine synthase, the enzyme responsible for the first step of hypusination (36), the possible effect of GC7 treatment on LHR mRNA downregulation was examined. Treatment with GC7 significantly reduced the intensity of upper band corresponding to hypusinated (hyp) eIF5A (band 1) with a corresponding increase in the intensity of the unhypusinated (pre) lower band (band 2) (Figure 3A). This was further confirmed using an in vitro hypusination assay as previously described (26, 29), using cytosolic extracts from the control (CTL) and downregulated (hCG) ovaries with or without GC7 pretreatment. The results (Figure 3B) showed that the incorporation of [3H]-spermidine into eIF5A was higher in the hCG treated samples (panel 1, lane 2) compared to control (panel 1, lane 1), and this increase was abrogated by GC7 pretreatment (panel 1, lane 3). These results showed that hCG treatment increases the levels of active/hypusinated form of eIF5A during LHR mRNA downregulation and this increase was abrogated by GC7 treatment. Inhibition of eIF5A activity by GC7 decreased the association of eIF5A with LHR mRNA and LRBP. Hypusination of eIF5A has been known to be essential for its function (23, 24), including its ability to bind RNA (25). Thus,


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hypusination inhibition by GC7 treatment is expected to

FIGURE 3. hCG increases eIF5A hypusination in rat ovaries during LHR downregulation. Day 5 superovulated rats were treated with GC7 (16 mg/kg b.w.; i.p.) on the fifth day of superovulation, followed by hCG (50 IU) 2h later. Rats were sacrificed and ovaries collected after 0, 4 and 6h and cytosolic extracts (S10) prepared as described in Methods. (A). Equal amounts of protein from the control (CTL) or hCG-treated S10 fractions with or without GC7 pretreatment were subjected to Western Blot analysis using eIF5A antibody (upper panel). The membranes were stripped and reprobed for ␤-tubulin (bottom panel). The blots shown are representative of three independent experiments. The graphs represent eIF5A levels normalized to ␤-tubulin and are shown as fold change vs. control. Error bars represent mean ⫾SE. *P ⬍ .05 vs. CTL and **P ⬍ .05 vs. hCG, n ⫽ 6. (B). Equal amounts of protein from the control (CTL) or hCG-treated S10 fractions with or without GC7 pretreatment were subjected to in vitro hypusination assay as described in the Methods section. The fluorography image is shown in the top panel. Western blot analyses of the same samples for eIF5A and tubulin are shown in the bottom panels. The blots shown are representative of three independent experiments. The graphs represent hyp-eIF5A levels normalized to ␤-tubulin and are shown as fold change vs. control. Error bars represent mean ⫾SE. *P ⬍ .05 vs. CTL and **P ⬍ .05 vs. hCG, n ⫽ 3.


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decrease the association of eIF5A with LHR mRNA and LRBP. Consistent with this notion, the results showed that inhibition of hypusination using GC7 abrogated the increases in the association of LHR mRNA to eIF5A as indicated by the RNA-IP and Real time PCR results presented in Figure 4A. HCG treatment increased the formation of eIF5A-LHR mRNA complex by 400% compared to control consistent with our earlier results (Figure 1B) whereas, GC7 pretreatment prevented this effect of hCG (Figure 4A). As a negative control, we showed that CYP11A1 mRNA, which is not regulated by LRBP showed no change in its association with eIF5A following

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hCG treatment (1.06-fold vs. CTL) or in response to GC7 pretreatment during the time period tested (1.35-fold vs. CTL; Figure 4B). This demonstrated that hCG-mediated increase in the association of LHR mRNA with hypusinated eIF5A is specific and related to LHR downregulation. Similarly, the increased binding of eIF5A and LRBP seen during hCG treatment was also completely inhibited by GC7 pretreatment as shown in Figure 4C, showing that LRBP can physically interact only with eIF5A in the hypusinated state. There was a 3.8-fold increase in the levels of LRBP bound to eIF5A following hCG treatment which was completely abrogated by GC7 treatment (GC7⫹hCG, 0.7- fold vs. CTL, P ⬍ .05). These results further supported the notion that hypusination is essential for the association of eIF5A with LHR mRNA as well as LRBP.

FIGURE 4. Inhibition of eIF5A hypusination using GC7 inhibits the association of eIF5A with LHR mRNA and LRBP. Rats were treated with GC7 (16 mg/kg b.w.; i.p.) on the fifth day of superovulation, followed by hCG (50 IU) 2h later. After sacrifice (6h after hCG), ovaries were collected and cytosolic extracts (S10) prepared as described in Methods. Equal amounts of protein from the control (CTL) or hCG-treated S10 fractions with or without GC7 pretreatment were subjected to RNA immunoprecipitation using antibodies against eIF5A followed by real time PCR using predesigned primers and probes for rat LH receptor mRNA (A) or CYP11A1 (B) as described in Methods. Total LHR mRNA present in the S10 fractions were also measured using real time PCR. The graphs in Figure 4A represent ratio of LHR mRNA levels in the immunoprecipitate normalized to total LHR mRNA and are shown as fold change vs. control. Error bars represent mean ⫾SE. *P ⬍ .05 vs. CTL and **P ⬍ .05 vs. hCG, n ⫽ 3. (C) Equal amounts of protein from the control (CTL) or hCG-treated S10 fractions with or without GC7 pretreatment were subjected to immunoprecipitation using eIF5A antibody. The immunoprecipitates were subjected to Western Blot analysis using LRBP antibody (upper panel). The membranes were stripped and reprobed for eIF5A (lower panel). The input was also analyzed for eIF5A, LRBP and ␤-tubulin (panels 3, 4 and 5). The blots shown are representative of three independent experiments. The graphs represent LRBP levels normalized to eIF5A and are shown as fold change vs. control. Error bars represent mean ⫾SE. *P ⬍ .05 vs. CTL and **P ⬍ .05 vs. hCG, n ⫽ 3.

Inhibition of eIF5A hypusination reduced ligand-induced downregulation of LH receptors. Since our data show that inhibition of eIF5A hypusination inhibited its association with LHR mRNA and LRBP, we extended this observation to determine if GC7 treatment would inhibit ligand-mediated LHR mRNA downregulation. To examine this, ovaries were harvested from control and hCG treated rats with or without GC7 pretreatment and LHR mRNA levels were analyzed using real time PCR. The results presented in Figure 5 show that the extent of LHR mRNA downregulation was significantly reduced by GC7 treatment. As expected, there was an 80% reduction in the levels of LHR mRNA after 8h of hCG treatment. When hypusination was inhibited by GC7 pretreatment, the extent of downregulation was significantly reduced. Since there was a significant reversal of downregulation of LHR mRNA expression by GC7, we then examined whether similar reversal also occurred in the expression of functional cell surface receptors. Membrane fractions were isolated from control and downregulated (8 and 12 hours) ovaries with or with-


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out GC7 pretreatment and the levels of functional receptors were analyzed using a specific 125I-hCG binding assay. As shown in Figures 6A and B, the binding of 125IhCG to membrane fraction was inhibited significantly, as expected, by 8h (Figure 6A) and 12h of downregulation by hCG treatment (Figure 6B) compared to control. There was a 50% decrease in the binding by 8h and 75% decrease by 12h post hCG treatment. Interestingly, pretreatment with GC7 completely abrogated the decrease seen at 8h and significantly reversed the decrease seen after 12 hours (Figure 6B). The kinetics of 125I-hCG binding to the LH receptors as a function of the ligand concentrations from CTL, hCG and GC7⫹hCG is presented in Figure 7. This binding data were then subjected to scatchard analysis (38) to determine the Kd and Bmax (Table 1). Three parallel lines were observed as shown in Figure 7 with

FIGURE 5. Pretreatment with GC7 inhibits LHR mRNA downregulation. Rats were treated with GC7 (16 mg/kg b.w.) on the fifth day of superovulation, followed by hCG (50 IU) 2h later. After sacrifice (8h after hCG), ovaries were collected and subjected to RNA isolation as described in Methods. RNAs were reverse transcribed, and the resulting cDNAs were subjected to real-time PCR quantitation using specific primers and probes for LHR mRNA and 18 sRNA. The graphs represent changes in LHR mRNA normalized to 18s rRNA and shown as fold change vs. control. Error bars represent mean ⫾ SE. *P ⬍ .05 vs. CTL and **P ⬍ .05 vs. hCG, n ⫽ 4.


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comparable Kd values. There was a significant decrease in the binding capacity (Bmax) in membrane fractions treated with hCG alone. As shown in Table 1, this decrease in Bmax in response to hCG treatment was reversed by GC7 treatment, without a significant change in Kd. These results further support that inhibition of hypusination by GC7 treatment significantly reversed hCG induced downregulation of LHR mRNA and 125I-hCG binding activity underscoring the role of eIF5A in the regulation of LHR expression in the ovary. Inhibition of eIF5A hypusination restored the downstream signaling by LH receptors during downregulation In order to analyze the presence of functional LH receptors on the cell surface, we examined one of its downstream targets, ERK 1/2. As shown in Figure 8, as expected, the downregulated (hCG, 8h) ovaries showed minimal ERK 1/2 phosphorylation (Panel 1, Lane 2) in response to treatment with hCG (1h), as expected, due to the loss of LH receptors. However, increased responsiveness was seen in the ovaries from animals pretreated with GC7 as evidenced by a robust hCG-mediated ERK1/2 phosphorylation (Panel 1, lane 3) which was comparable to the control ovaries treated with hCG (Panel 1, lane 4). This further confirmed the reversal of downregulation and restoration of functional cell surface LH receptors by inhibiting hypusination of eIF5A. These results clearly show that hypusinated eIF5A plays a crucial role in LHR downregulation in the ovary.

Discussion

The post-transcriptional regulation of LH receptor mRNA is most evident during the downregulation of LHR in response to preovulatory LH surge. Our previous studies have shown that this occurs predominantly through the participation of an LHR mRNA binding protein (LRBP) (8 –10, 39). The current study focused on the role of eukaryotic initiation factor 5A (eIF5A) in LHR downregulation through its association with LRBP-LHR mRNA FIGURE 6. Inhibition of eIF5A hypusination using GC7 significantly reversed the complex. Our present results prohCG-induced decrease in the number of cell surface LH receptors. Rats were vide strong evidence to support the treated with GC7 (16 mg/kg b.w.) on the fifth day of superovulation, followed by hCG (50 IU) 2h notion that the levels of hypusinated later. Ovaries were collected 0, 8 and 12 hours later and membrane fractions prepared as described in Methods. Equal amounts of protein from the control (CTL) or hCG-treated eIF5A increase during hCG-induced membrane fractions with or without GC7 pretreatment were incubated with 125I-hCG and the downregulation and that the hypusibound receptor expression was calculated as described in Methods. Binding is expressed as cpm/ nated eIF5A then associates with ␮g protein. Error bars represent mean ⫾SE. *P ⬍ .05 vs. CTL and **P ⬍ .05 vs. hCG, n ⫽ 3.


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Table 1. Binding constants of CTL, hCG and GC7⫹hCG treated samples.

CTL hCG GC7⫹hCG

Bmax (fmol/␮g DNA) 0.83 0.19 0.65

K (nM) 2.2 2.6 2.1 d

The Bmax and Kd values were calculated using the binding data from Fig 7, as described in Methods.

LHR mRNA-LRBP complex in the ribosomes. Furthermore, the inhibition of hypusination significantly reduces the association of eIF5A with LHR mRNA as well as LRBP and significantly reverses the downregulation of LHR mRNA causing restoration of ligand binding activity and downstream signaling events. In our earlier studies using yeast two hybrid screen, eIF5A was identified as one of the proteins that interacted with LRBP during LHR downregulation. eIF5A is a small (17-kDa) protein that is highly conserved throughout evolution (40). In its active state, eIF5A has been shown to contain the unique polyamine-derived amino acid hypusine (41). Hypusine (N⑀-[4-amino-2-hydroxybutyl]lysine) is formed post-translationally in a reaction involving modification of the residue Lys50 of eIF5A by the sequential action of enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH), and is necessary for many eIF5A functions (for review see ref

FIGURE 7. Scatchard analysis of the binding of 125I-hCG to the membrane fractions. Rats were treated with GC7 2h prior to injection with hCG on the fifth day of pseudopregnancy, ovaries were collected 0 and 12 hours later and membrane fractions prepared from control (CTL), hCG, and GC7⫹hCG treated rats. Functional receptor expression was measured by calculating its binding to different concentrations of 125I-hCG in a binding assay, as described in detail in Methods. Each point represents average of three determinations for total binding and two determinations for nonspecific binding. A representative result of three different independent experiments is shown. Figure shows the transformation of the binding data to scatchard plot to calculate Bmax and Kd.

(23).). eIF5A was first isolated from rabbit reticulocyte lysate ribosomes and classified as an initiation factor (17, 42– 44), but later studies suggested that it plays a more ubiquitous role in cells including translational elongation (19 –21), cellular proliferation (45, 46), apoptosis (47– 49) and nucleocytoplasmic transport (15, 16). Furthermore, it is shown to be involved in various aspects of RNA metabolism including mRNA degradation and turnover (17, 18). The interaction of eIF5A with RNA in vitro depends on the presence of deoxyhypusine or hypusine moiety in eIF5A (25, 50). Since LHR mRNA downregulation has been shown to be regulated by increased LHR mRNA degradation, we hypothesized that eIF5A might play a role through its interaction with LRBP and LHR mRNA. The results presented here conclusively demonstrate that hypusination of eIF5A increases during hCG-induced downregulation of LHR mRNA. We further show that inhibition of hypusination of eIF5A by pretreatment with GC7 abrogated the association of eIF5A with the LRBP-LHR mRNP complex blocking the extent of LHR mRNA downregulation as evidenced by the restoration of LH receptor expression and LH-mediated signaling. This is consistent with the recently demonstrated role of eIF5A hypusination in the direct post-

FIGURE 8. Inhibition of eIF5A hypusination restores loss of downstream signaling during LHR downregulation. Rats were treated with GC7 2h prior to injection with hCG (50 IU) on the fifth day of pseudopregnancy. After 8h, they were given another dose of hCG (50 IU). Ovaries were collected 1 hour later and cytosolic extracts (S10) prepared using RIPA buffer. Equal amounts of protein from the control (CTL) or hCG-treated S10 fractions were subjected to Western Blot analysis using p- ERK1/2 antibody. The membranes were stripped and reprobed for total ERK 2 (lower panel). The blots shown are representative of three independent experiments. The graphs represent changes in p-ERK1/2 levels normalized to total ERK2 levels and shown as fold change vs. control. Error bars represent mean ⫾ SE. *P ⬍ .05 vs. CTL (- hCG) and **P ⬍ .05 vs. hCG (⫹ hCG), n ⫽ 3.


doi: 10.1210/me.2014-1132

transcriptional regulation of iNOS (Nos2) mRNA in both rodent and human cells (37). The polyamine-dependent post-transcriptional regulation of the cancer-related gene, cyclooxygenase-2 (COX-2), which contains eIF-5A consensus binding elements, was also shown to be mediated through eIF5A (51). We also found that the downregulation of functional LH receptors on the membrane fraction and its reversal by GC7 occurs in a manner similar to that seen for LHR mRNA downregulation. Regulation of LH receptor mRNA levels plays an important role in controlling the responsiveness of the luteal cells to gonadotropins. There is a close correlation between LH receptor mRNA expression levels and ligand binding activity (30, 52) further suggesting that loss of hCG binding seen during hCGinduced downregulation, at least in major part, is due to the loss of LHR mRNA. However, the inhibition of hCG binding was less pronounced (Figure 6B) than the inhibition observed in the decrease of mRNA levels (Figure 5). This could be due to the shorter half-life of the mRNA compared tothat of the protein. eIF5A has also been shown to be involved in formation of P-bodies, which are sites of mRNA decay (53), as well as function as a nucleocytoplasmic shuttle protein (54). We have shown that LHR mRNA-LRBP complex is rerouted from ribosomes to P-bodies for degradation during downregulation (11). In this context, we examined the potential role of eIF5A as a shuttle protein facilitating the translocation of LRBP-LHR mRNA complex to the Pbodies. Since were unable to find an increase in the association eIF5A and the P-body marker DCP1A during downregulation (supplemental figure, Figure S1), it does not appear that eIF5A elicits its effect through translocation of LHR mRNA to the P-bodies. Thus, eIF5A might play a role augmenting translational suppression of LHR mRNA-LRBP complex prior to reaching the degradation site. This is supported by the fact that there is increased association of eIF5A with the LRBP-LHR mRNP complex in the translating polysomes. However, the association of eIF5A with LRBP does not appear to be bridged by LHR mRNA. It is likely that LRBP has distinct binding sites for both eIF5A and LHR mRNA. The results also suggest that LRBP associates with LHR mRNA and eIF5A independent of each other, to form an untranslatable complex in the ribosomes, which is then targeted for degradation. Currently, it is unclear whether eIF5A directly recognizes LHR mRNA or the interaction occurs through the association of LHR mRNA with LRBP. Further studies are needed to address this issue. However, our immunoprecipitation experiments clearly show that LHR mRNA is part of the LRBP-eIF5A complex. In conclusion, the present study provides strong evi-


9

dence for the role of eIF5A in LRBP-mediated degradation of LH receptor mRNA. Based on our present results and previous findings (10, 11, 34, 39, 55), a schematic summary of the mechanism of LHR downregulation including the participation of eIF5A is presented in Figure 9. LH surge or a pharmacological dose of hCG induces the LHR-mediated activation of cyclic AMP production leading to an increase in LRBP expression and association

FIGURE 9. Schematic model depicting the proposed role of hypusinated eIF5A in LRBP-mediated LHR mRNA downregulation. Binding of LH/hCG to LH receptor stimulates the expression of LRBP and its LHR mRNA binding activity. LH/hCG treatment also increased the levels of hypusinated eIF5A, which then forms a complex with LRBP-LHR mRNA leading to the degradation of LHR mRNA.


10


with LHR mRNA. hCG treatment also leads to increase in the levels of hypusinated eIF5A culminating in the degradation of LHR mRNA. This study therefore shed light on the previously unrecognized role of eIF5A in the acute regulation of LHR, a member of the G protein coupled receptor family.

Mol Endocrinol

11.

12.

Acknowledgments

13.

This work was supported by National Institutes of Health Grant R01 HD 06656 –37. We thank Helle Peegel and Jennifer White Sinden for technical assistance and Dr. Aaron Goldstrohm for his many helpful suggestions and critical evaluation of the manuscript.

14.

Received April 24, 2014. Accepted September 8, 2014. *Address all correspondence and requests for reprints to: K.M.J. Menon, 6428 Medical Sciences Building I, 1301 Catherine Street, Ann Arbor, MI 48109 – 0617; Fax: ⫹1 734 –9368617, Tel: ⫹1 734 –764-8142; E-mail: [email protected]. This work was supported by Grant Support: R01 HD0665637. Disclosure summary: The authors have nothing to disclose. 1 BM and TG contributed equally to this work.

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