Regulation of Luteinizing Hormone/Chorionic Gonadotropin Receptor Messenger Ribonucleic Acid Expression in the Rat Ovary: Relationship to Ch
http://www.100md.com
内分泌学杂志 2005年第1期
Departments of Obstetrics and Gynecology and Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109
Address all correspondence and requests for reprints to: K. M. J. Menon, 6428 Medical Science I, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0617. E-mail: kmjmenon@umich.edu.
Abstract
Down-regulation of LH/human chorionic gonadotropin (hCG) receptor (LHR) mRNA in the ovary after the preovulatory LH surge or the administration of a pharmacological dose of LH/hCG occurs through a posttranscriptional mechanism. A LHR mRNA-binding protein was identified as the LHR mRNA destabilizing factor, and its identity was established as mevalonate kinase (Mvk). In the present study, we determined that, in the pseudopregnant rat ovary, LHR mRNA levels began to fall 4 h after hCG injection, at which time Mvk protein levels were elevated, and this elevation was preceded by an increase in Mvk mRNA levels. When the cytosolic fractions of hCG-treated ovaries were subjected to RNA EMSA, an increase in LHR mRNA-LHR mRNA-binding protein complex formation was observed, in parallel with the increase of Mvk expression. We also found that hCG coordinately up-regulated the expression of Mvk and other sterol-responsive elements containing cholesterol biosynthesis enzymes, such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and farnesyl pyrophosphate synthase. This up-regulation was transient, but the hCG-induced ovarian cholesterol depletion lasted for more than 24 h. Taken together, our results suggest that, in the ovary, LH/hCG up-regulates the expression of cholesterol biosynthesis enzymes and lipoprotein receptors to replenish cellular cholesterol, and the up-regulation of Mvk leads to a down-regulation of LHR and suppresses the LH/hCG signal cascade transiently. Thus Mvk, an enzyme involved in cholesterol biosynthesis, serves as a link between LHR mRNA expression and cellular cholesterol metabolism.
Introduction
THE LUTEINIZING/HUMAN chorionic gonadotropin (hCG) receptor (LHR), a member of Gs protein receptor family, is expressed in the gonads (1, 2). Like other members of this family, LHR expression is down-regulated by exposure to a pharmacological dose of its ligand (3). It has been shown that LHR expression is regulated during the ovarian cycle under normal physiological conditions. For example, in response to the LH surge before ovulation, LHR undergoes transient down-regulation followed by restoration to normal preexisting levels within 72 h (3, 4, 5, 6). Previous studies have demonstrated that the decline in ovarian cell surface LHR induced by LH surge or administration of a pharmacological dose of hCG is paralleled by a marked loss of all four LHR mRNA transcripts, and this loss is not due to a decrease in transcription but rather to increased degradation (3, 5). A specific RNA-binding protein was identified in the rat ovary, which was found to bind to a cytidine-rich region of LHR mRNA (4, 7) and accelerate its degradation in vitro (8). Amino-terminal analysis and MS-MALDI of the gel purified LHR mRNA-binding protein (LRBP) established its identity as mevalonate kinase (Mvk) (9). The binding characteristics of the recombinant Mvk for LHR mRNA recognition are similar to those seen for the endogenous LRBP isolated from rat ovary (4, 7, 9).
Mvk, an enzyme involved in cholesterol biosynthesis, was first cloned from rat liver (10, 11). Rat Mvk contains 395 amino acids with a deduced molecular mass of 42 kDa. Although plasma-derived cholesterol plays a major role as a substrate for steroid synthesis (12, 13) through receptor mediated uptake, e.g. low-density lipoprotein (LDL) and high-density lipoprotein (HDL) (14, 15), de novo synthesized cholesterol is also used as a precursor for ovarian steroid hormone production. A common feature of the genes encoding LDL receptor (LDLR), HDL receptor (HDLR), and the enzymes involved in cholesterol biosynthesis is the presence of a sterol regulatory element (SRE), and their expression is regulated by SRE-binding proteins (SREBPs), the transcription factors activated by hCG-induced cholesterol depletion (16, 17). The promoter regions of Mvk, as well as other enzymes in the mevalonate pathway, including 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (HMGS) and HMG-CoA reductase (HMGR), also contain SREs (16, 17, 18). However, the regulatory mechanism of Mvk expression in the ovary, either as a cholesterol biosynthesis enzyme or as an RNA-binding protein, has not been examined.
The present studies were carried out to examine the changes of Mvk expression in relation to its LHR mRNA binding activity during hCG-induced down-regulation. We have also examined the changes in the expression of other key cholesterol biosynthesis enzymes and lipoprotein receptors in the context of ovarian cholesterol metabolism.
Materials and Methods
Animals and treatments
All animals were under the care of the University of Michigan Unit of Laboratory Animal Medicine. Pseudopregnancy was induced in 22-d-old Sprague Dawley female rats by a sc injection of 50 IU pregnant mare serum gonadotropin (PMSG) (Calbiochem, La Jolla, CA), followed by 25 IU hCG (Sigma, St. Louis, MO) 56 h later. The day of hCG injection was taken as d 0. On the fifth day of pseudopregnancy, rats were injected with 50 IU hCG to induce LH receptor down-regulation; control rats received saline (8). Ovaries were collected at time intervals as indicated in specific experiments.
Isolation of total RNA
Tissues, collected at each time interval, were pooled from two animals before homogenization. Total RNA was extracted using the commercial product TRIzol Reagent (Invitrogen, Carlsbad, CA) with a modified TRI reagent procedure (19).
Rapid amplification of 3' cDNA ends (3'RACE)
The cDNA was synthesized using the SuperScript III First Strand Synthesis System for RT-PCR kit (Invitrogen). In a total vol of 20 μl, 1 μg total RNA was incubated with 200 U SuperScript III reverse transcriptase and 50 pmol adaptor primer (5'-CCG CTC GAG ACT GAT CAA CTA CG (T)15-3'), at 50 C for 50 min. The reaction was stopped by heating to 85 C for 10 min, and 1 μl first-strand product was amplified in 50 μl Platinum PCR SuperMix High Fidelity (Invitrogen) plus 10 pmol universal amplification primer (UAP) (5'-CCG CTC GAG ACT GAT CAA CTA CG-3') and 10 pmol gene-specific primer (GSP) (5'-CGC GGA TCC ATG TTG TCA GAA GTC CTG-3') (Fig. 1A). After 35 cycles (denaturation at 94 C for 30 sec, annealing at 55 C for 40 sec, extension at 72 C for 2 min, and a final extension at 72 C for 5 min after the last cycle) of amplification on a DNA Engine Peltier Thermal Cycler (MJ Research, Waltham, MA), 10 μl RACE product was fractioned in 1% agarose gel and stained with ethidium bromide, as previously described (20).
FIG. 1. Analysis of Mvk mRNA transcript in rat ovary. A, Schematic structure of the rat liver Mvk mRNA, and positions and sequences of primers used. B, Ethidium bromide-stained gel showing the products of 3'RACE and nested amplification. Total RNA from hCG down-regulated rat ovary (lane 1), control rat ovary (lane 2), and control rat liver (lane 3) were subjected to 3'RACE. 3'RACE products of hCG down-regulated ovary (lane 4), control ovary (lane 5), and liver (lane 6) were further nested-amplified. C, Ethidium bromide-stained gel showing the fragments of 3'RACE products from hCG down-regulated ovary (lane 1), control ovary (lane 2), and liver (lane 3) after MseI digestion. UTR, Untranslated region; M.W., molecular weight.
Nested amplification and restriction digestion
The 3'RACE products were gel-extracted, using the Montage Gel Extraction kit (Millipore, Billerica, MA), and used as template for a nested amplification, with 10 pmol UAP and 10 pmol nested GSP (5'-CCGCAGAGCAATGGGAAAGTG-3'). The gel-extracted 3'RACE products were also subjected to digestion by restriction enzymes, and results were examined by fractionation on 2% agarose gel.
Probe construction
The Mvk cDNA probe was prepared by RT-PCR, using total RNA extracted from the pseudopregnant rat ovary as template. The sense strand corresponded to nucleotides 82–1269 of the Mvk cDNA (10) and was cloned into a pcDNA4/HisMax vector between the BamHI and XhoI restriction sites. Sequence of the probe was verified by dideoxy chain termination sequencing. The LHR cDNA probe has been described earlier (5).
Northern blot analysis
Aliquots of total RNA were separated by electrophoresis in 1.2% agarose-formaldehyde gels and transferred to nitrocellulose membranes. Blots were subjected to UV cross-linking and prehybridized at 42 C for 2 h in a solution containing 0.5 mg/ml salmon sperm DNA and 2x hybridization buffer [1.5 M NaCl-0.1 M TES (N-tris[hydroxymethyl]methyl-2-aminoethene sulfonic acid (pH 7.1)-0.1 M EDTA-2x Denhardt’s solution] diluted 1:1 with deionized formamide. Probes were radiolabeled with [-32P] deoxycycidine triphosphate (ICN, Costa Mesa, CA) and hybridized to blots overnight at 42 C in fresh hybridization buffer. Hybridized blots were washed five times with 2x saline sodium citrate containing 0.1% sodium dodecyl sulfate and then exposed to Kodak x-ray film (Eastman Kodak, Rochester, NY) at –80 C or subjected to analysis by the PhosphorImager (Bio-Rad, Hercules, CA).
Preparation of cytosolic proteins
Pooled ovaries were homogenized in buffer A (10 mM HEPES, pH7.9; 0.5 mM MgCl2; 50 μM EDTA; 5 mM dithiothreitol; and 10% glycerol) containing 50 mM KCl and EDTA-free protease inhibitor mixture (4). The homogenates were centrifuged at 105,000 x g for 90 min at 4 C, and the supernatants (S-100) were quantified using BCA Protein Assay kit (Pierce, Rockford, IL).
RNA EMSA (REMSA)
REMSA was performed as described previously from our laboratory (8). In brief, the S100 extracts from control or down-regulated rat ovaries were incubated with 1 x 105 cpm radiolabeled gel-purified RNA (4) in homogenization buffer A, for 10 min at 30 C, in the presence of 5 μg tRNA and 40 U RNasin (Promega, Madison, WI), Unprotected radiolabeled RNA was degraded by addition of 2 U ribonuclease T1, and the RNA-protein complexes were then resolved by 5% native PAGE at 4 C. The gel was dried and exposed to Kodak x-ray film.
Western blot analysis
Cytosolic protein samples (30 μg) were denatured by boiling for 5 min in the presence of loading buffer and were subjected to electrophoresis on a 10% Laemmli SDS-PAGE. After electrophoresis, the proteins were electroblotted onto nitrocellulose membranes (0.2-μm pore) and blocked overnight at 4 C. The membranes were then incubated for 1 h with antibodies at room temperature. Mvk antibody, which was raised against the N-terminal amino acids, was generously provided by Dr. Skaidrite K. Krisans, San Diego State University (21). ?-Tubulin antibody was a commercial product (Sigma). The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody for 45 min at room temperature, and signals were visualized by the enhanced chemiluminescence system (Amersham, Piscataway, NJ).
Real-time PCR
Total RNAs (250 ng) were reverse transcribed in a vol of 50 μl, using 125 pmol random hexamer, 500 μM deoxynucleotide triphosphate, and 62.5 U MultiScribe reverse transcriptase (PE Applied Biosystems, Foster City, CA). The resulting cDNAs were diluted to 250 μl with ribonuclease free water. Each real-time PCR consisted of 5 μl cDNA template, 12.5 μl 1x SYBR Green PCR Master Mix (PE Applied Biosystems), and 10 pmol forward and reverse primers in a final vol of 25 μl. Reactions were carried out on an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems) for 40 cycles (95 C for 15 sec, 60 C for 1 min) after initial 10 min of incubation at 95 C. The primers used for real-time PCR are listed in Table 1. Amplicon size and reaction specificity were confirmed by agarose gel electrophoresis. The fold change in expression of each gene was calculated using the standard curve method, with the ribosomal protein (RP) S16 as internal control (22, 23).
TABLE 1. Primers used for real-time PCR
Cholesterol determination
Pooled ovaries were weighed and homogenized with 10 vol homogenization buffer (0.3 M sucrose, 25 mM Tris-HCl, and 1 mM EDTA, pH 7.4). Lipid extraction was performed using the method of Bligh and Dyer (24), with the addition of 20,000 cpm [3H] cholesteryl oleate and 20,000 cpm [14C] cholesterol to monitor recovery. After extraction with chloroform/methanol, the chloroform layer was collected and evaporated at 45 C under a gentle stream of nitrogen. The pellet was dissolved in 500 μl isopropanol and assayed for total and free cholesterol by the enzymatic assay described earlier (25). Briefly, 1 ml working reagent (6 mM sodium cholate, 1.5% Triton X-100, 7.5 mM phenol, 0.5 mM 4-aminoanitipyrine, 16.4 U peroxidase, and 0.08 U cholesterol oxidase) was added to 40 μl of each standard or unknown sample, and the absorbance was measured at 500 nm. For total cholesterol determination, the samples were hydrolyzed with 1.6 U cholesterol esterase to convert esterified cholesterol to free cholesterol, and the resulting total cholesterol was assayed by the enzymatic method described above. During the procedure, recovery of cholesterol and cholesterol ester was more than 80%, on the basis of the recovery of [3H] and [14C] as monitored by added [3H] and [14C] sterols.
Statistical analysis
Statistical analysis was carried out using the unpaired t test, with Sigma Stat software (version 2.0; SPSS Inc., Chicago IL). Each experiment was repeated at least three times with similar results. Blots are representative of one experiment, and graphs represent the mean ± SEM of at least three experiments.
Results
Analysis of Mvk mRNA transcript in rat ovary
Previous studies revealed that there is a marked difference in the activity and kinetic properties between ovarian Mvk and liver Mvk, raising the possibility that an Mvk isoform(s) may exist in the ovary (26). Therefore, before examining the regulation of Mvk mRNA expression in pseudopregnant rat ovary, attempts were made to determine whether ovarian Mvk mRNA is expressed as a single transcript similar to that seen in liver (10).
Total RNA was extracted from ovaries of both control and hCG-treated rats, and for comparison, RNA was also extracted from liver. These RNA samples were reverse transcribed using an adaptor primer, which contains oligo- deoxythymidylate and a UAP sequence (Fig. 1A). The N-terminal amino acids of purified LRBP had been determined previously (MLSEVLLVSA) (9). Based on this sequence, a GSP1 was designed. Using primers GSP1 and UAP, a single fragment, approximately 1.7 kb, was amplified from both control and hCG-treated rat ovaries, whereas a similar band and two additional smaller bands, most likely artifacts, were seen in the control rat liver (Fig. 1B, lanes 1–3).
To confirm authenticity of the 3'RACE products, bands corresponding to approximately1.7 kb were extracted from each lane. They were used as template for nested amplification, and a single band, approximately 1.6-kb, was detected in all cases (Fig. 1B, lanes 4–6). The 3'RACE products were also subjected to enzymatic cleavage by Mse I, a restriction enzyme known to cleave liver Mvk mRNA at positions 254 and 635 bp (10). Fragments of expected sizes (170 bp, 380 bp, and 1.1 kb) were obtained from each of the 3'RACE products, and the digestion patterns were essentially the same (Fig. 1C). Enzymatic digestions by AvaI and StuI were conducted, and confirmatory results were obtained (data not shown). From these experiments, we conclude that a single form of Mvk mRNA exists in both hormone-treated and control ovaries, and it is identical with the reported liver Mvk mRNA (10).
Changes in LHR and Mvk mRNA levels in the hCG down-regulated rat ovary
Previous studies from our laboratory demonstrated that the loss of LHR mRNA during hCG-induced down-regulation was due to accelerated mRNA decay, and a transacting protein was involved in this process (4, 7, 8, 9). As indicated in the introduction, the protein has been identified as Mvk, which binds to a cytidine-rich region of LHR mRNA, and the binding was shown to be regulated by hCG (4, 8). To extend the study further, we examined regulation of Mvk expression by hCG. On d 5 of pseudopregnancy, rats were treated with 50 IU hCG (or equal volume of saline), and the LHR and Mvk mRNA levels were measured in ovaries collected at 12, 24, and 48 h. Results presented in Fig. 2A showed, as expected, a concomitant decline of all four LHR mRNA transcripts after hCG injection and a gradual increase of these transcripts in control rats. However, Mvk mRNA expression did not show a discernible increase during these time intervals (Fig. 2B). The intensity of Mvk mRNA, quantified in densitometric units and normalized to 18S rRNA (Fig. 2D), revealed a parallel increase of Mvk mRNA in both control and hCG-treated samples.
FIG. 2. Effect of hCG on LHR and Mvk mRNA expression in pesudopregnant rat ovaries (0–48 h). Rats were injected with hCG or saline on the fifth day of pseudopregnancy, and ovaries were collected at 0, 12, 24, and 48 h. A–C, Autoradiograph of Northern blot of rat LHR mRNA, Mvk mRNA, and 18S rRNA. Twenty micrograms of total RNA from individual samples were fractionated on agarose gel, transferred to membrane, hybridized to probes, and exposed to x-ray film. The Northern blot is representative of two independent experiments. D, Quantification of Mvk mRNA levels. The blot was scanned, and corresponding bands were quantitated using NIH image 1.61 software. Data were normalized for the amount of 18S rRNA in each sample and expressed relative to the value at time zero. Each point represents the average ± SE of three separate densitometric scans. Statistical differences between the time zero time control value and different time intervals are indicated by the symbol () (P < 0.05).
Considering the fact that the majority of LHR mRNA (>90%) is degraded within 12 h after hCG injection, we measured the changes of LHR and Mvk mRNA levels during this period by Northern blot analysis (Fig. 3). A decline in the LHR mRNA expression was seen at 4 h, and it was preceded by a marked increase of Mvk mRNA expression. In fact, the elevation of Mvk mRNA was seen immediately after hCG injection and ended before the 6-h time interval. To quantitate the changes in mRNA levels more accurately, the same samples were subjected to mRNA quantification by real-time PCR. Two sets of primers were designed for LHR, and one set of primers was designed for Mvk. RP S16 mRNA (22, 27, 28, 29) was used as an internal control (Table 1). Real-time PCR results showed that there was a significant increase in Mvk mRNA expression starting at 2 h and a return to control level by 6 h, which is consistent with the Northern blot data (see Fig. 6, A–C). From these results, we conclude that hCG-induced decline in LHR mRNA levels is preceded by an increase in Mvk mRNA expression.
FIG. 3. Effect of hCG on LHR and Mvk mRNA expression in pseudopregnant rat ovaries (0–12 h). Rats were injected with hCG or saline on the fifth day of pseudopregnancy, and ovaries were collected at 0, 1, 2, 4, 6, and 12 h. A, Phosphoimage of Northern blot of rat LHR mRNA, Mvk mRNA, and 18S rRNA. Twenty micrograms of total RNA from individual samples were fractionated on agarose gel, transferred to membrane, hybridized to probes, and subjected to PhosphoImager scanning. B, Quantification of Mvk mRNA levels. Phosphoimages were quantified using Bio-Rad Quantity One software. The value for Mvk in each sample was normalized to the signal obtained for 18S rRNA, and the data are expressed as percent of RNA at time zero. Each point represents the average ± SE of three independent experiments. Mvk mRNA levels in hCG-treated ovaries show significant increase. Statistical differences between treated and control samples are indicated by the symbol (*) (P < 0.05).
FIG. 6. Effect of hCG on specific mRNA expression in pseudopregnant rat ovaries (0–12 h). Rats were injected with hCG or saline on the fifth day of pseudopregnancy, and ovaries were collected at 0, 2, 4, 6, and 12 h. A–C, Measurement of steady-state levels of LHR (using primer set LHR and primer set LHR 2) and Mvk mRNAs, by real-time PCR. Mean values ± SE (n = 3) were normalized to RP S16 and graphed as percent of control (time 0 h). D–H, Measurement of steady-state levels of HMGS, HMGR, FPPS, HDLR, and LDLR mRNAs by real-time PCR. Mean values ± SE (n = 3) were normalized to RP S16 and graphed as percent of control (time 0 h). Statistical differences between treated and control samples are indicated by the symbol (*) (P < 0.05).
Changes in Mvk protein levels and LHR mRNA binding activity during hCG-induced down-regulation
Experiments were designed to determine whether the increase in Mvk mRNA was also reflected at the protein level. In addition, we examined whether the increase in Mvk protein level produced a parallel increase in LHR mRNA binding activity. Pseudopregnant rats were injected with 50 IU hCG or saline (control) on the fifth day. Ovaries were collected at 2, 4, 6, and 12 h, and S100 fractions were prepared as described under Materials and Methods. Western blot analysis was done using an antibody against the first 15 N-terminal amino acids of Mvk (MLSEVLLVSAPGKVI) (21). As shown in Fig. 4, a prominent 42-kDa band, corresponding to the size of Mvk, was seen on the gel, and its intensity started to increase at 4 h and remained elevated up to 12 h, in the down-regulated ovaries but not in the controls. Additionally, a faint band, most likely an artifact, appeared under the major 42-kDa band in each lane, and its intensity increased along with the major band (Fig. 4). The same batch of S100 samples was subjected to REMSA to determine the LHR mRNA binding activity. The results showed that, after hCG injection, the LHR mRNA binding activity in the S100 fractions started to increase at 4 h and remained high at 6 h and 12 h (Fig. 5). These findings indicate that, during hCG- induced down-regulation, LHR mRNA binding activity increases in a manner comparable with that of Mvk protein levels. Furthermore, both increases were preceded by the increase in Mvk mRNA expression.
FIG. 4. Regulation of Mvk protein level by hCG in the pseudopregnant rat ovary (0–12 h). Rats were injected with hCG or saline on the fifth day of pseudopregnancy, and ovaries were collected at 0, 2, 4, 6, and 12 h. A, Immunoblot of cytosolic proteins with antibodies against Mvk and ?-tubulin. B, Quantification of Mvk protein levels. The blots were scanned, and 42-kDa bands were quantified using NIH image 1.61 software. Data were normalized for the amount of ?-tubulin in each sample and expressed relative to the value at time zero. Each point represents the average ± SE of three independent experiments. Statistical differences between treated and control samples are indicated by the symbol (*) (P < 0.05).
FIG. 5. Effect of hCG on the LRBP/Mvk binding activity in pseudopregnant rat ovaries (0–12 h). Rats were injected with hCG or saline on the fifth day of pseudopregnancy, and ovaries were collected at 0, 2, 4, 6, and 12 h. A, Autoradiograph of REMSA performed for measuring LRBP/Mvk binding activity. Fifty micrograms of cytosolic proteins collected at each time point were incubated with 1 x 105 cpm radiolabeled RNA. The RNA-protein complexes were resolved by native polyacrylamide gel and exposed to x-ray film. B, Quantification of LRBP/Mvk binding activity. Radiolabeled bands were quantified using NIH image 1.61 software. The data are expressed relative to the value at time zero. Each point represents the average ± SE of three independent experiments. Statistical differences between treated and control samples are indicated by the symbol (*) (P < 0.05).
Effect of hCG on mRNA levels of sterol-responsive genes in the pseudopregnant rat ovary
In ovary, cholesterol, the substrate for steroidogenesis, is derived from both de novo biosynthesis and receptor-mediated uptake via plasma lipoproteins, LDL and HDL (12, 13). The de novo synthesis pathway of cholesterol includes a series of enzymatic reactions, involving HMGS, HMGR, Mvk, and farnesyl pyrophosphate synthase (FPPS). Because the regulatory mechanisms for the expression of these genes are complex, involving both cAMP- and SREBP-dependent pathways (17, 30, 31, 32), we examined whether their expressions are coordinately regulated, under conditions in which LHR mRNA is down-regulated by hCG. Ovaries from control and hCG-treated animals were collected at 2, 4, 6, and 12 h, and mRNA levels of HMGS, HMGR, and FPPS were measured by real-time PCR. Results presented in Fig. 6, D–F, show that, upon treatment with hCG, the expression of all three genes was up-regulated in a coordinated manner, reaching maximum at 4 h, and declined afterward. These results indicate that Mvk, a LHR mRNA destabilizing factor, is coordinately regulated with other cholesterol biosynthesis enzymes at the transcriptional level in response to hCG treatment.
Because the ovary derives cholesterol mainly from plasma LDL and HDL (12, 13, 15), LDLR and HDLR mRNA levels were examined to extrapolate further the relationship among cholesterol transport, cholesterol biosynthesis, and LHR mRNA expression. HCG injection caused a persistent increase in LDLR and HDLR mRNA levels, both of which remained elevated at 12 h. (Fig. 6, G–H). The time-courses of LDLR and HDLR mRNA expression were different from those of cholesterol biosynthesis enzymes (Fig. 6, D–F), which suggests that, although lipoprotein receptors, like the cholesterol biosynthesis enzymes, are up-regulated by hCG at the transcriptional level, the mechanisms of regulation are probably different.
Effect of hCG on ovarian sterol concentrations
It is known that the gene expression of cholesterol biosynthesis enzymes and plasma lipoprotein receptors is negatively regulated in cholesterol-rich environment, via a SREBP-dependent pathway (33, 34, 35). Thus, the effect of hCG administration on the cholesterol content in pseudopregnant rat ovaries was examined in relation to the coordinate up-regulation of Mvk and related proteins. The ovarian cholesterol levels were assayed at 2, 4, 12, 18, and 24 h, after hCG injection to d-5 pseudopregnant rats. The results presented in Fig. 7A show that, as expected, treatment with hCG caused a rapid decline in the total cellular cholesterol level, which reached its lowest level at 4 h and remained low even at 24 h, In contrast, the cholesterol content in control ovaries was unchanged over the period. Free cholesterol measurements followed a pattern similar to that seen with total cholesterol (Fig. 7B). These results indicate that, during the 24-h period after hCG down-regulation, the ovarian cholesterol content remains depleted, which explains the continuous high levels of LDLR and HDLR mRNAs.
FIG. 7. Regulation of total/free cholesterol levels by hCG in pseudopregnant rat ovary (0–24 h). Rats were injected with hCG or saline on the fifth day of pseudopregnancy, and ovaries were collected at 0, 2, 4, 12, 18, and 24 h. Data are expressed as the mean of three determinations ± SE. Statistical differences between treated and control samples are indicated by the symbol (*) (P < 0.05).
Discussion
Previous studies have shown that LHR mRNA down-regulation in response to a pharmacological dose of hCG is mediated by a specific LRBP (4, 7, 8). The identity of LRBP was established as Mvk, an enzyme involved in cholesterol biosynthesis (9). Furthermore, both ATP and mevalonate competed for the binding of Mvk to LHR mRNA, suggesting that the catalytic site of Mvk might participate in the binding of LHR mRNA either directly or indirectly (9). Structural studies of Mvk using bacterially expressed enzyme revealed its similarity to the GHMP family of proteins (36), which possess a ?--? loop known as ribosomal protein S5 domain 2-like fold similar to that found in proteins that interact with rRNA (9). These facts strongly support the notion that Mvk mediates LHR mRNA degradation by binding to the mRNA molecule specifically.
Although the presence of Mvk in the liver of rat and pig was demonstrated in the late 1950s (37), the cloning (10), purification (11), and structure/function relationship (38, 39, 40) of purified enzyme have been accomplished only more recently. Despite the extensive studies in the liver, only limited studies have been carried out in nonhepatic tissues (26). As a prelude to the study on the regulation of Mvk in ovary, we performed 3'RACE and determined that Mvk mRNA is present in a single molecular form in the ovary, which is identical with that found in the liver. Furthermore, Western blot analysis was conducted using an antibody against the N-terminal amino acids of Mvk (21). Though multiple immunoreactive bands were detected using ovarian samples (Fig. 4) as well as liver samples, the 42-kDa band appeared to be the predominant one, which is consistent with the report of Biardi et al. (21). In liver, Biardi et al. detected multiple bands using this antibody, but a single 42-kDa band was detected using antibodies against different epitopes of Mvk. Thus, we believe that there is only one 42-kDa Mvk protein in rat ovary, and other bands are artifacts.
After establishing that the mRNA transcript of Mvk in ovary is identical with that found in liver, the effects of hCG treatment on its expression and function were determined in the context of the down-regulation of LHR mRNA expression. Our results showed that hCG-induced down-regulation of LHR mRNA was demonstrable at 4 h after hormone treatment, and this loss of mRNA was coincident with an increase in LHR mRNA binding activity of Mvk from the S-100 fraction. Western blot results also showed a corresponding increase, suggesting that the increased LHR mRNA binding is a result of elevated Mvk protein content in the cytosolic fractions of the ovarian cells. This elevation was preceded by a short-term increase of Mvk mRNA levels, as demonstrated by both Northern blot (Fig. 3) and real-time PCR data (Fig. 6). The persistence of Mvk protein level beyond time periods when its mRNA level had returned to the control level suggests that the decay rate of protein is slower than that of mRNA. An alternate explanation is that hCG treatment may have triggered other events, such as increased translation or protein stabilization. Our data indicated that hCG-induced elevation of Mvk mRNA levels lasted for a short duration, starting immediately after hCG injection and ending within a few hours. In fact, after 12 h, Mvk mRNA expression levels showed an increase in both hCG-treated and control rat ovaries. Thus, the onset of the loss of steady-state levels of LHR mRNA appears to closely correlate with the early effect of hCG treatment on Mvk expression.
In the ovary, cholesterol required for steroidogenesis and other cellular functions is acquired through de novo synthesis and through receptor-mediated uptake from LDL and HDL (30, 41, 42). The receptors for LDL and HDL as well as HMGR in ovary have been shown to be induced by LH/hCG (30, 43, 44). Because cholesterol transport/synthesis and steroidogenesis in the ovary is intimately associated with LHR expression, the involvement of Mvk as a protein that regulates LHR mRNA expression is highly significant. Our results, using real-time PCR, demonstrate that hCG not only stimulated the expression of Mvk leading to accelerated LHR mRNA decay but also increased the expression of a number of sterol-responsive genes that contribute to cholesterol accumulation in the ovary (Fig. 8). So far, this is the first report showing that, in pseudopregnant rat ovary, expression of cholesterol biosynthesis enzymes and lipoprotein receptors are coordinately up-regulated by hCG. Increased expression of ovarian Mvk, together with other enzymes such as HMGS and HMGR, is expected to contribute to cellular cholesterol accumulation, although it is presently unclear as to how Mvk serves its dual functions, as an enzyme and an RNA-binding protein. Whether there is a factor that is required for switching Mvk’s function from a catalytic protein to an RNA-binding protein remains an open question at this time.
FIG. 8. Proposed model of LHR down-regulation by LH/hCG in luteinized ovarian cells. LH/hCG binding to the LHR activates the cAMP signal cascade and increases the transcription of a series of cholesterol biosynthesis enzymes and steroidogenesis. An increase in Mvk protein level was seen 4 h after hCG injection. This leads to an increase in Mvk-LHR mRNA complex formation and accelerates LHR mRNA decay. The decrease of LHR mRNA level culminates in the loss of cell surface LHR and produces a transient abolishment of the signal cascade. PP, Pyrophosphate.
It should be noticed that, whereas the expression of cholesterol biosynthesis enzymes returned to control level within a few hours, the expression of LDLR and HDLR mRNA remains high up to 12 h after hCG treatment. It is interesting to note that the cellular cholesterol levels remain low in hCG-treated animals compared with the control (Fig. 7). This may point out the different mechanisms by which plasma lipoprotein receptors and cholesterol biosynthesis enzymes are regulated. Lopez et al. (17) reported that, in PMSG-primed rats, hCG treatment induces cholesterol depletion, leading to increased production of sterol regulatory element-binding protein-1a (SREBP-1a). This factor and SF-1, a transcription factor activated directly by cAMP, synergize the induction of HDLR expression (17). Our present results, that the expression of HDL and LDR remained high at a time when cell surface LHR was completely down-regulated, are consistent with this notion. The rapid depletion of ovarian cholesterol after hCG injection suggests that cellular cholesterol status may be a factor that plays a role in increasing Mvk expression. However, the rapid decrease of Mvk mRNA levels between 4–6 h, despite the cholesterol depletion prevailing for more than 24 h, cannot explain this simple relationship. Although we have not examined the direct transcriptional regulation of Mvk by cAMP, available evidence (45) points to such a mechanism.
In summary, we present evidence for a novel mechanism for the regulation of LHR mRNA expression in the ovary. Our results show that hCG stimulates Mvk expression, which leads to increased binding to LHR mRNA and accelerates its decay. Furthermore, hCG coordinately up-regulates the expression of Mvk, along with other cholesterol biosynthesis enzymes and lipoprotein receptors. (Fig. 8).
Acknowledgments
The authors express their appreciation to Dr. Anil K Nair, Christine Clouser, Helle Peegel, Dr. Roberto Towns, and Dr. Pradeep Kayampilly for critical reading of the manuscript.
References
Ascoli M, Fanelli F, Segaloff DL 2002 The lutropin/choriogonadotropin receptor, a 2002 perspective. Endocr Rev 23:141–174
Menon KMJ, Munshi UM, Clouser CL, Nair AK 2004 Regulation of luteinizing hormone/human chorionic gonadotropin receptor expression: a perspective. Biol Reprod 70:861–866
Hoffman YM, Peegel H, Sprock MJ, Zhang QY, Menon KMJ 1991 Evidence that human chorionic gonadotropin/luteinizing hormone receptor down-regulation involves decreased levels of receptor messenger ribonucleic acid. Endocrinology 128:388–393
Kash JC, Menon KMJ 1998 Identification of a hormonally regulated luteinizing hormone/human chorionic gonadotropin receptor mRNA binding protein. Increased mRNA binding during receptor down-regulation. J Biol Chem 273:10658–10664
Lu DL, Peegel H, Mosier SM, Menon KMJ 1993 Loss of lutropin/human choriogonadotropin receptor messenger ribonucleic acid during ligand-induced down-regulation occurs post transcriptionally. Endocrinology 132:235–240
LaPolt PS, Oikawa M, Jia XC, Dargan C, Hsueh AJ 1990 Gonadotropin-induced up- and down-regulation of rat ovarian LH receptor message levels during follicular growth, ovulation and luteinization. Endocrinology 126:3277–3279
Kash JC, Menon KMJ 1999 Sequence-specific binding of a hormonally regulated mRNA binding protein to cytidine-rich sequences in the lutropin receptor open reading frame. Biochemistry 38:16889–16897
Nair AK, Kash JC, Peegel H, Menon KMJ 2002 Post-transcriptional regulation of luteinizing hormone receptor mRNA in the ovary by a novel mRNA-binding protein. J Biol Chem 277:21468–21473
Nair AK, Menon KMJ 2004 Isolation and characterization of a novel trans-factor for luteinizing hormone receptor mRNA from ovary. J Biol Chem 279:14937–14944
Tanaka RD, Lee LY, Schafer BL, Kratunis VJ, Mohler WA, Robinson GW, Mosley ST 1990 Molecular cloning of mevalonate kinase and regulation of its mRNA levels in rat liver. Proc Natl Acad Sci USA 87:2872–2876
Tanaka RD, Schafer BL, Lee LY, Freudenberger JS, Mosley ST 1990 Purification and regulation of mevalonate kinase from rat liver. J Biol Chem 265:2391–2398
Gwynne JT, Strauss III JF 1982 The role of lipoproteins in steroidogenesis and cholesterol metabolism in steroidogenic glands. Endocr Rev 3:299–329
Azhar S, Reaven E 2002 Scavenger receptor class BI and selective cholesteryl ester uptake: partners in the regulation of steroidogenesis. Mol Cell Endocrinol 195:1–26
Li X, Peegel H, Menon KMJ 1998 In situ hybridization of high density lipoprotein (scavenger, type 1) receptor messenger ribonucleic acid (mRNA) during folliculogenesis and luteinization: evidence for mRNA expression and induction by human chorionic gonadotropin specifically in cell types that use cholesterol for steroidogenesis. Endocrinology 139:3043–3049
Hwang J, Menon KMJ 1983 Characterization of low density and high density lipoprotein receptors in the rat corpus luteum and regulation by gonadotropin. J Biol Chem 258:8020–8027
Bishop RW, Chambliss KL, Hoffmann GF, Tanaka RD, Gibson KM 1998 Characterization of the mevalonate kinase 5'-untranslated region provides evidence for coordinate regulation of cholesterol biosynthesis. Biochem Biophys Res Commun 242:518–524
Lopez D, McLean MP 1999 Sterol regulatory element-binding protein-1a binds to cis elements in the promoter of the rat high density lipoprotein receptor SR-BI gene. Endocrinology 140:5669–5681
Edwards PA, Ericsson J 1998 Signaling molecules derived from the cholesterol biosynthetic pathway: mechanisms of action and possible roles in human disease. Curr Opin Lipidol 9:433–440
Chomczynski P, Mackey K 1995 Short technical reports. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. Biotechniques 19:942–945
Wang L, Xie LP, Huang WQ, Yao B, Pu RL, Zhang RQ 2001 Presence of gonadotropin-releasing hormone (GnRH) and its mRNA in rat pancreas. Mol Cell Endocrinol 172:185–191
Biardi L, Sreedhar A, Zokaei A, Vartak NB, Bozeat RL, Shackelford JE, Keller GA, Krisans SK 1994 Mevalonate kinase is predominantly localized in peroxisomes and is defective in patients with peroxisome deficiency disorders. J Biol Chem 269:1197–1205
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F 2002 Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034
Chan YL, Paz V, Olvera J, Wool IG 1990 The primary structure of rat ribosomal protein S16. FEBS Lett 263:85–88
Bligh EG, Dyer WJ 1959 A rapid method of total lipid extraction and purification. Can J Med Sci 37:911–917
Li X, Peegel H, Menon KMJ 2001 Regulation of high density lipoprotein receptor messenger ribonucleic acid expression and cholesterol transport in theca-interstitial cells by insulin and human chorionic gonadotropin. Endocrinology 142:174–181
Flint AP 1970 The activity and kinetic properties of mevalonate kinase in superovulated rat ovary. Biochem J 120:145–150
Leo CP, Pisarska MD, Hsueh AJ 2001 DNA array analysis of changes in preovulatory gene expression in the rat ovary. Biol Reprod 65:269–276
Bowen JM, Telleria CM, Towns R, Keyes PL 2000 Down-regulation of long-form prolactin receptor mRNA during prolactin-induced luteal regression. Eur J Endocrinol 143:285–292
Dasmahapatra AK, Trewin AL, Hutz RJ 2002 Estrous cycle-regulated expression of CYP1B1 mRNA in the rat ovary. Comp Biochem Physiol B Biochem Mol Biol 133:127–134
Golos TG, Strauss III JF 1987 Regulation of low density lipoprotein receptor gene expression in cultured human granulosa cells: roles of human chorionic gonadotropin, 8-bromo-3',5'-cyclic adenosine monophosphate, and protein synthesis. Mol Endocrinol 1:321–326
Wood JR, Strauss III JF 2002 Multiple signal transduction pathways regulate ovarian steroidogenesis. Rev Endocr Metab Disord 3:33–46
Horton JD, Goldstein JL, Brown MS 2002 SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 109:1125–1131[Free Full Text]
Goldstein JL, Brown MS 1990 Regulation of the mevalonate pathway. Nature 343:425–430
Brown MS, Goldstein JL 1999 A proteolytic pathway that control the cholesterol content of membranes, cells, and blood. Proc Natl Acad Sci USA 96:11041–11048
Brown MS, Goldstein JL 1997 The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89:331–340
Krepkiy D, Miziorko HM 2004 Identification of active site residues in mevalonate diphosphate decarboxylase: implications for a family of phosphotransferases. Protein Sci 13:1875–1881
Levy HR, Popjak G 1960 Studies on the biosynthesis of cholesterol. 10. Mevalonic kinase from liver. Biochem J 75:417–428
Potter D, Wojnar JM, Narasimhan C, Miziorko HM 1997 Identification and functional characterization of an active-site lysine in mevalonate kinase. J Biol Chem 272:5741–5746
Potter D, Miziorko HM 1997 Identification of catalytic residues in human mevalonate kinase. J Biol Chem 272:25449–25454
Fu Z, Wang M, Potter D, Miziorko HM, Kim JJ 2002 The structure of a binary complex between a mammalian mevalonate kinase and ATP: insights into the reaction mechanism and human inherited disease. J Biol Chem 277:18134–18142
Rajendran KG, Hwang J, Menon KMJ 1983 Binding, degradation, and utilization of plasma high density and low density lipoproteins for progesterone production in cultured rat luteal cells. Endocrinology 112:1746–1753
Azhar S, Nomoto A, Leers-Sucheta S, Reaven E 1998 Simultaneous induction of an HDL receptor protein (SR-BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell model. J Lipid Res 39:1616–1628
Reaven E, Nomoto A, Leers-Sucheta S, Temel R, Williams DL, Azhar S 1998 Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian models. Endocrinology 139:2847–2856
Azhar S, Chen YD, Reaven GM 1984 Gonadotropin modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in desensitized luteinized rat ovary. Biochemistry 23:4533–4538
Medicherla S, Azhar S, Cooper A, Reaven E 1996 Regulation of cholesterol responsive genes in ovary cells: impact of cholesterol delivery systems. Biochemistry 35:6243–6250(Lei Wang and K. M. J. Men)
Address all correspondence and requests for reprints to: K. M. J. Menon, 6428 Medical Science I, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0617. E-mail: kmjmenon@umich.edu.
Abstract
Down-regulation of LH/human chorionic gonadotropin (hCG) receptor (LHR) mRNA in the ovary after the preovulatory LH surge or the administration of a pharmacological dose of LH/hCG occurs through a posttranscriptional mechanism. A LHR mRNA-binding protein was identified as the LHR mRNA destabilizing factor, and its identity was established as mevalonate kinase (Mvk). In the present study, we determined that, in the pseudopregnant rat ovary, LHR mRNA levels began to fall 4 h after hCG injection, at which time Mvk protein levels were elevated, and this elevation was preceded by an increase in Mvk mRNA levels. When the cytosolic fractions of hCG-treated ovaries were subjected to RNA EMSA, an increase in LHR mRNA-LHR mRNA-binding protein complex formation was observed, in parallel with the increase of Mvk expression. We also found that hCG coordinately up-regulated the expression of Mvk and other sterol-responsive elements containing cholesterol biosynthesis enzymes, such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and farnesyl pyrophosphate synthase. This up-regulation was transient, but the hCG-induced ovarian cholesterol depletion lasted for more than 24 h. Taken together, our results suggest that, in the ovary, LH/hCG up-regulates the expression of cholesterol biosynthesis enzymes and lipoprotein receptors to replenish cellular cholesterol, and the up-regulation of Mvk leads to a down-regulation of LHR and suppresses the LH/hCG signal cascade transiently. Thus Mvk, an enzyme involved in cholesterol biosynthesis, serves as a link between LHR mRNA expression and cellular cholesterol metabolism.
Introduction
THE LUTEINIZING/HUMAN chorionic gonadotropin (hCG) receptor (LHR), a member of Gs protein receptor family, is expressed in the gonads (1, 2). Like other members of this family, LHR expression is down-regulated by exposure to a pharmacological dose of its ligand (3). It has been shown that LHR expression is regulated during the ovarian cycle under normal physiological conditions. For example, in response to the LH surge before ovulation, LHR undergoes transient down-regulation followed by restoration to normal preexisting levels within 72 h (3, 4, 5, 6). Previous studies have demonstrated that the decline in ovarian cell surface LHR induced by LH surge or administration of a pharmacological dose of hCG is paralleled by a marked loss of all four LHR mRNA transcripts, and this loss is not due to a decrease in transcription but rather to increased degradation (3, 5). A specific RNA-binding protein was identified in the rat ovary, which was found to bind to a cytidine-rich region of LHR mRNA (4, 7) and accelerate its degradation in vitro (8). Amino-terminal analysis and MS-MALDI of the gel purified LHR mRNA-binding protein (LRBP) established its identity as mevalonate kinase (Mvk) (9). The binding characteristics of the recombinant Mvk for LHR mRNA recognition are similar to those seen for the endogenous LRBP isolated from rat ovary (4, 7, 9).
Mvk, an enzyme involved in cholesterol biosynthesis, was first cloned from rat liver (10, 11). Rat Mvk contains 395 amino acids with a deduced molecular mass of 42 kDa. Although plasma-derived cholesterol plays a major role as a substrate for steroid synthesis (12, 13) through receptor mediated uptake, e.g. low-density lipoprotein (LDL) and high-density lipoprotein (HDL) (14, 15), de novo synthesized cholesterol is also used as a precursor for ovarian steroid hormone production. A common feature of the genes encoding LDL receptor (LDLR), HDL receptor (HDLR), and the enzymes involved in cholesterol biosynthesis is the presence of a sterol regulatory element (SRE), and their expression is regulated by SRE-binding proteins (SREBPs), the transcription factors activated by hCG-induced cholesterol depletion (16, 17). The promoter regions of Mvk, as well as other enzymes in the mevalonate pathway, including 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (HMGS) and HMG-CoA reductase (HMGR), also contain SREs (16, 17, 18). However, the regulatory mechanism of Mvk expression in the ovary, either as a cholesterol biosynthesis enzyme or as an RNA-binding protein, has not been examined.
The present studies were carried out to examine the changes of Mvk expression in relation to its LHR mRNA binding activity during hCG-induced down-regulation. We have also examined the changes in the expression of other key cholesterol biosynthesis enzymes and lipoprotein receptors in the context of ovarian cholesterol metabolism.
Materials and Methods
Animals and treatments
All animals were under the care of the University of Michigan Unit of Laboratory Animal Medicine. Pseudopregnancy was induced in 22-d-old Sprague Dawley female rats by a sc injection of 50 IU pregnant mare serum gonadotropin (PMSG) (Calbiochem, La Jolla, CA), followed by 25 IU hCG (Sigma, St. Louis, MO) 56 h later. The day of hCG injection was taken as d 0. On the fifth day of pseudopregnancy, rats were injected with 50 IU hCG to induce LH receptor down-regulation; control rats received saline (8). Ovaries were collected at time intervals as indicated in specific experiments.
Isolation of total RNA
Tissues, collected at each time interval, were pooled from two animals before homogenization. Total RNA was extracted using the commercial product TRIzol Reagent (Invitrogen, Carlsbad, CA) with a modified TRI reagent procedure (19).
Rapid amplification of 3' cDNA ends (3'RACE)
The cDNA was synthesized using the SuperScript III First Strand Synthesis System for RT-PCR kit (Invitrogen). In a total vol of 20 μl, 1 μg total RNA was incubated with 200 U SuperScript III reverse transcriptase and 50 pmol adaptor primer (5'-CCG CTC GAG ACT GAT CAA CTA CG (T)15-3'), at 50 C for 50 min. The reaction was stopped by heating to 85 C for 10 min, and 1 μl first-strand product was amplified in 50 μl Platinum PCR SuperMix High Fidelity (Invitrogen) plus 10 pmol universal amplification primer (UAP) (5'-CCG CTC GAG ACT GAT CAA CTA CG-3') and 10 pmol gene-specific primer (GSP) (5'-CGC GGA TCC ATG TTG TCA GAA GTC CTG-3') (Fig. 1A). After 35 cycles (denaturation at 94 C for 30 sec, annealing at 55 C for 40 sec, extension at 72 C for 2 min, and a final extension at 72 C for 5 min after the last cycle) of amplification on a DNA Engine Peltier Thermal Cycler (MJ Research, Waltham, MA), 10 μl RACE product was fractioned in 1% agarose gel and stained with ethidium bromide, as previously described (20).
FIG. 1. Analysis of Mvk mRNA transcript in rat ovary. A, Schematic structure of the rat liver Mvk mRNA, and positions and sequences of primers used. B, Ethidium bromide-stained gel showing the products of 3'RACE and nested amplification. Total RNA from hCG down-regulated rat ovary (lane 1), control rat ovary (lane 2), and control rat liver (lane 3) were subjected to 3'RACE. 3'RACE products of hCG down-regulated ovary (lane 4), control ovary (lane 5), and liver (lane 6) were further nested-amplified. C, Ethidium bromide-stained gel showing the fragments of 3'RACE products from hCG down-regulated ovary (lane 1), control ovary (lane 2), and liver (lane 3) after MseI digestion. UTR, Untranslated region; M.W., molecular weight.
Nested amplification and restriction digestion
The 3'RACE products were gel-extracted, using the Montage Gel Extraction kit (Millipore, Billerica, MA), and used as template for a nested amplification, with 10 pmol UAP and 10 pmol nested GSP (5'-CCGCAGAGCAATGGGAAAGTG-3'). The gel-extracted 3'RACE products were also subjected to digestion by restriction enzymes, and results were examined by fractionation on 2% agarose gel.
Probe construction
The Mvk cDNA probe was prepared by RT-PCR, using total RNA extracted from the pseudopregnant rat ovary as template. The sense strand corresponded to nucleotides 82–1269 of the Mvk cDNA (10) and was cloned into a pcDNA4/HisMax vector between the BamHI and XhoI restriction sites. Sequence of the probe was verified by dideoxy chain termination sequencing. The LHR cDNA probe has been described earlier (5).
Northern blot analysis
Aliquots of total RNA were separated by electrophoresis in 1.2% agarose-formaldehyde gels and transferred to nitrocellulose membranes. Blots were subjected to UV cross-linking and prehybridized at 42 C for 2 h in a solution containing 0.5 mg/ml salmon sperm DNA and 2x hybridization buffer [1.5 M NaCl-0.1 M TES (N-tris[hydroxymethyl]methyl-2-aminoethene sulfonic acid (pH 7.1)-0.1 M EDTA-2x Denhardt’s solution] diluted 1:1 with deionized formamide. Probes were radiolabeled with [-32P] deoxycycidine triphosphate (ICN, Costa Mesa, CA) and hybridized to blots overnight at 42 C in fresh hybridization buffer. Hybridized blots were washed five times with 2x saline sodium citrate containing 0.1% sodium dodecyl sulfate and then exposed to Kodak x-ray film (Eastman Kodak, Rochester, NY) at –80 C or subjected to analysis by the PhosphorImager (Bio-Rad, Hercules, CA).
Preparation of cytosolic proteins
Pooled ovaries were homogenized in buffer A (10 mM HEPES, pH7.9; 0.5 mM MgCl2; 50 μM EDTA; 5 mM dithiothreitol; and 10% glycerol) containing 50 mM KCl and EDTA-free protease inhibitor mixture (4). The homogenates were centrifuged at 105,000 x g for 90 min at 4 C, and the supernatants (S-100) were quantified using BCA Protein Assay kit (Pierce, Rockford, IL).
RNA EMSA (REMSA)
REMSA was performed as described previously from our laboratory (8). In brief, the S100 extracts from control or down-regulated rat ovaries were incubated with 1 x 105 cpm radiolabeled gel-purified RNA (4) in homogenization buffer A, for 10 min at 30 C, in the presence of 5 μg tRNA and 40 U RNasin (Promega, Madison, WI), Unprotected radiolabeled RNA was degraded by addition of 2 U ribonuclease T1, and the RNA-protein complexes were then resolved by 5% native PAGE at 4 C. The gel was dried and exposed to Kodak x-ray film.
Western blot analysis
Cytosolic protein samples (30 μg) were denatured by boiling for 5 min in the presence of loading buffer and were subjected to electrophoresis on a 10% Laemmli SDS-PAGE. After electrophoresis, the proteins were electroblotted onto nitrocellulose membranes (0.2-μm pore) and blocked overnight at 4 C. The membranes were then incubated for 1 h with antibodies at room temperature. Mvk antibody, which was raised against the N-terminal amino acids, was generously provided by Dr. Skaidrite K. Krisans, San Diego State University (21). ?-Tubulin antibody was a commercial product (Sigma). The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody for 45 min at room temperature, and signals were visualized by the enhanced chemiluminescence system (Amersham, Piscataway, NJ).
Real-time PCR
Total RNAs (250 ng) were reverse transcribed in a vol of 50 μl, using 125 pmol random hexamer, 500 μM deoxynucleotide triphosphate, and 62.5 U MultiScribe reverse transcriptase (PE Applied Biosystems, Foster City, CA). The resulting cDNAs were diluted to 250 μl with ribonuclease free water. Each real-time PCR consisted of 5 μl cDNA template, 12.5 μl 1x SYBR Green PCR Master Mix (PE Applied Biosystems), and 10 pmol forward and reverse primers in a final vol of 25 μl. Reactions were carried out on an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems) for 40 cycles (95 C for 15 sec, 60 C for 1 min) after initial 10 min of incubation at 95 C. The primers used for real-time PCR are listed in Table 1. Amplicon size and reaction specificity were confirmed by agarose gel electrophoresis. The fold change in expression of each gene was calculated using the standard curve method, with the ribosomal protein (RP) S16 as internal control (22, 23).
TABLE 1. Primers used for real-time PCR
Cholesterol determination
Pooled ovaries were weighed and homogenized with 10 vol homogenization buffer (0.3 M sucrose, 25 mM Tris-HCl, and 1 mM EDTA, pH 7.4). Lipid extraction was performed using the method of Bligh and Dyer (24), with the addition of 20,000 cpm [3H] cholesteryl oleate and 20,000 cpm [14C] cholesterol to monitor recovery. After extraction with chloroform/methanol, the chloroform layer was collected and evaporated at 45 C under a gentle stream of nitrogen. The pellet was dissolved in 500 μl isopropanol and assayed for total and free cholesterol by the enzymatic assay described earlier (25). Briefly, 1 ml working reagent (6 mM sodium cholate, 1.5% Triton X-100, 7.5 mM phenol, 0.5 mM 4-aminoanitipyrine, 16.4 U peroxidase, and 0.08 U cholesterol oxidase) was added to 40 μl of each standard or unknown sample, and the absorbance was measured at 500 nm. For total cholesterol determination, the samples were hydrolyzed with 1.6 U cholesterol esterase to convert esterified cholesterol to free cholesterol, and the resulting total cholesterol was assayed by the enzymatic method described above. During the procedure, recovery of cholesterol and cholesterol ester was more than 80%, on the basis of the recovery of [3H] and [14C] as monitored by added [3H] and [14C] sterols.
Statistical analysis
Statistical analysis was carried out using the unpaired t test, with Sigma Stat software (version 2.0; SPSS Inc., Chicago IL). Each experiment was repeated at least three times with similar results. Blots are representative of one experiment, and graphs represent the mean ± SEM of at least three experiments.
Results
Analysis of Mvk mRNA transcript in rat ovary
Previous studies revealed that there is a marked difference in the activity and kinetic properties between ovarian Mvk and liver Mvk, raising the possibility that an Mvk isoform(s) may exist in the ovary (26). Therefore, before examining the regulation of Mvk mRNA expression in pseudopregnant rat ovary, attempts were made to determine whether ovarian Mvk mRNA is expressed as a single transcript similar to that seen in liver (10).
Total RNA was extracted from ovaries of both control and hCG-treated rats, and for comparison, RNA was also extracted from liver. These RNA samples were reverse transcribed using an adaptor primer, which contains oligo- deoxythymidylate and a UAP sequence (Fig. 1A). The N-terminal amino acids of purified LRBP had been determined previously (MLSEVLLVSA) (9). Based on this sequence, a GSP1 was designed. Using primers GSP1 and UAP, a single fragment, approximately 1.7 kb, was amplified from both control and hCG-treated rat ovaries, whereas a similar band and two additional smaller bands, most likely artifacts, were seen in the control rat liver (Fig. 1B, lanes 1–3).
To confirm authenticity of the 3'RACE products, bands corresponding to approximately1.7 kb were extracted from each lane. They were used as template for nested amplification, and a single band, approximately 1.6-kb, was detected in all cases (Fig. 1B, lanes 4–6). The 3'RACE products were also subjected to enzymatic cleavage by Mse I, a restriction enzyme known to cleave liver Mvk mRNA at positions 254 and 635 bp (10). Fragments of expected sizes (170 bp, 380 bp, and 1.1 kb) were obtained from each of the 3'RACE products, and the digestion patterns were essentially the same (Fig. 1C). Enzymatic digestions by AvaI and StuI were conducted, and confirmatory results were obtained (data not shown). From these experiments, we conclude that a single form of Mvk mRNA exists in both hormone-treated and control ovaries, and it is identical with the reported liver Mvk mRNA (10).
Changes in LHR and Mvk mRNA levels in the hCG down-regulated rat ovary
Previous studies from our laboratory demonstrated that the loss of LHR mRNA during hCG-induced down-regulation was due to accelerated mRNA decay, and a transacting protein was involved in this process (4, 7, 8, 9). As indicated in the introduction, the protein has been identified as Mvk, which binds to a cytidine-rich region of LHR mRNA, and the binding was shown to be regulated by hCG (4, 8). To extend the study further, we examined regulation of Mvk expression by hCG. On d 5 of pseudopregnancy, rats were treated with 50 IU hCG (or equal volume of saline), and the LHR and Mvk mRNA levels were measured in ovaries collected at 12, 24, and 48 h. Results presented in Fig. 2A showed, as expected, a concomitant decline of all four LHR mRNA transcripts after hCG injection and a gradual increase of these transcripts in control rats. However, Mvk mRNA expression did not show a discernible increase during these time intervals (Fig. 2B). The intensity of Mvk mRNA, quantified in densitometric units and normalized to 18S rRNA (Fig. 2D), revealed a parallel increase of Mvk mRNA in both control and hCG-treated samples.
FIG. 2. Effect of hCG on LHR and Mvk mRNA expression in pesudopregnant rat ovaries (0–48 h). Rats were injected with hCG or saline on the fifth day of pseudopregnancy, and ovaries were collected at 0, 12, 24, and 48 h. A–C, Autoradiograph of Northern blot of rat LHR mRNA, Mvk mRNA, and 18S rRNA. Twenty micrograms of total RNA from individual samples were fractionated on agarose gel, transferred to membrane, hybridized to probes, and exposed to x-ray film. The Northern blot is representative of two independent experiments. D, Quantification of Mvk mRNA levels. The blot was scanned, and corresponding bands were quantitated using NIH image 1.61 software. Data were normalized for the amount of 18S rRNA in each sample and expressed relative to the value at time zero. Each point represents the average ± SE of three separate densitometric scans. Statistical differences between the time zero time control value and different time intervals are indicated by the symbol () (P < 0.05).
Considering the fact that the majority of LHR mRNA (>90%) is degraded within 12 h after hCG injection, we measured the changes of LHR and Mvk mRNA levels during this period by Northern blot analysis (Fig. 3). A decline in the LHR mRNA expression was seen at 4 h, and it was preceded by a marked increase of Mvk mRNA expression. In fact, the elevation of Mvk mRNA was seen immediately after hCG injection and ended before the 6-h time interval. To quantitate the changes in mRNA levels more accurately, the same samples were subjected to mRNA quantification by real-time PCR. Two sets of primers were designed for LHR, and one set of primers was designed for Mvk. RP S16 mRNA (22, 27, 28, 29) was used as an internal control (Table 1). Real-time PCR results showed that there was a significant increase in Mvk mRNA expression starting at 2 h and a return to control level by 6 h, which is consistent with the Northern blot data (see Fig. 6, A–C). From these results, we conclude that hCG-induced decline in LHR mRNA levels is preceded by an increase in Mvk mRNA expression.
FIG. 3. Effect of hCG on LHR and Mvk mRNA expression in pseudopregnant rat ovaries (0–12 h). Rats were injected with hCG or saline on the fifth day of pseudopregnancy, and ovaries were collected at 0, 1, 2, 4, 6, and 12 h. A, Phosphoimage of Northern blot of rat LHR mRNA, Mvk mRNA, and 18S rRNA. Twenty micrograms of total RNA from individual samples were fractionated on agarose gel, transferred to membrane, hybridized to probes, and subjected to PhosphoImager scanning. B, Quantification of Mvk mRNA levels. Phosphoimages were quantified using Bio-Rad Quantity One software. The value for Mvk in each sample was normalized to the signal obtained for 18S rRNA, and the data are expressed as percent of RNA at time zero. Each point represents the average ± SE of three independent experiments. Mvk mRNA levels in hCG-treated ovaries show significant increase. Statistical differences between treated and control samples are indicated by the symbol (*) (P < 0.05).
FIG. 6. Effect of hCG on specific mRNA expression in pseudopregnant rat ovaries (0–12 h). Rats were injected with hCG or saline on the fifth day of pseudopregnancy, and ovaries were collected at 0, 2, 4, 6, and 12 h. A–C, Measurement of steady-state levels of LHR (using primer set LHR and primer set LHR 2) and Mvk mRNAs, by real-time PCR. Mean values ± SE (n = 3) were normalized to RP S16 and graphed as percent of control (time 0 h). D–H, Measurement of steady-state levels of HMGS, HMGR, FPPS, HDLR, and LDLR mRNAs by real-time PCR. Mean values ± SE (n = 3) were normalized to RP S16 and graphed as percent of control (time 0 h). Statistical differences between treated and control samples are indicated by the symbol (*) (P < 0.05).
Changes in Mvk protein levels and LHR mRNA binding activity during hCG-induced down-regulation
Experiments were designed to determine whether the increase in Mvk mRNA was also reflected at the protein level. In addition, we examined whether the increase in Mvk protein level produced a parallel increase in LHR mRNA binding activity. Pseudopregnant rats were injected with 50 IU hCG or saline (control) on the fifth day. Ovaries were collected at 2, 4, 6, and 12 h, and S100 fractions were prepared as described under Materials and Methods. Western blot analysis was done using an antibody against the first 15 N-terminal amino acids of Mvk (MLSEVLLVSAPGKVI) (21). As shown in Fig. 4, a prominent 42-kDa band, corresponding to the size of Mvk, was seen on the gel, and its intensity started to increase at 4 h and remained elevated up to 12 h, in the down-regulated ovaries but not in the controls. Additionally, a faint band, most likely an artifact, appeared under the major 42-kDa band in each lane, and its intensity increased along with the major band (Fig. 4). The same batch of S100 samples was subjected to REMSA to determine the LHR mRNA binding activity. The results showed that, after hCG injection, the LHR mRNA binding activity in the S100 fractions started to increase at 4 h and remained high at 6 h and 12 h (Fig. 5). These findings indicate that, during hCG- induced down-regulation, LHR mRNA binding activity increases in a manner comparable with that of Mvk protein levels. Furthermore, both increases were preceded by the increase in Mvk mRNA expression.
FIG. 4. Regulation of Mvk protein level by hCG in the pseudopregnant rat ovary (0–12 h). Rats were injected with hCG or saline on the fifth day of pseudopregnancy, and ovaries were collected at 0, 2, 4, 6, and 12 h. A, Immunoblot of cytosolic proteins with antibodies against Mvk and ?-tubulin. B, Quantification of Mvk protein levels. The blots were scanned, and 42-kDa bands were quantified using NIH image 1.61 software. Data were normalized for the amount of ?-tubulin in each sample and expressed relative to the value at time zero. Each point represents the average ± SE of three independent experiments. Statistical differences between treated and control samples are indicated by the symbol (*) (P < 0.05).
FIG. 5. Effect of hCG on the LRBP/Mvk binding activity in pseudopregnant rat ovaries (0–12 h). Rats were injected with hCG or saline on the fifth day of pseudopregnancy, and ovaries were collected at 0, 2, 4, 6, and 12 h. A, Autoradiograph of REMSA performed for measuring LRBP/Mvk binding activity. Fifty micrograms of cytosolic proteins collected at each time point were incubated with 1 x 105 cpm radiolabeled RNA. The RNA-protein complexes were resolved by native polyacrylamide gel and exposed to x-ray film. B, Quantification of LRBP/Mvk binding activity. Radiolabeled bands were quantified using NIH image 1.61 software. The data are expressed relative to the value at time zero. Each point represents the average ± SE of three independent experiments. Statistical differences between treated and control samples are indicated by the symbol (*) (P < 0.05).
Effect of hCG on mRNA levels of sterol-responsive genes in the pseudopregnant rat ovary
In ovary, cholesterol, the substrate for steroidogenesis, is derived from both de novo biosynthesis and receptor-mediated uptake via plasma lipoproteins, LDL and HDL (12, 13). The de novo synthesis pathway of cholesterol includes a series of enzymatic reactions, involving HMGS, HMGR, Mvk, and farnesyl pyrophosphate synthase (FPPS). Because the regulatory mechanisms for the expression of these genes are complex, involving both cAMP- and SREBP-dependent pathways (17, 30, 31, 32), we examined whether their expressions are coordinately regulated, under conditions in which LHR mRNA is down-regulated by hCG. Ovaries from control and hCG-treated animals were collected at 2, 4, 6, and 12 h, and mRNA levels of HMGS, HMGR, and FPPS were measured by real-time PCR. Results presented in Fig. 6, D–F, show that, upon treatment with hCG, the expression of all three genes was up-regulated in a coordinated manner, reaching maximum at 4 h, and declined afterward. These results indicate that Mvk, a LHR mRNA destabilizing factor, is coordinately regulated with other cholesterol biosynthesis enzymes at the transcriptional level in response to hCG treatment.
Because the ovary derives cholesterol mainly from plasma LDL and HDL (12, 13, 15), LDLR and HDLR mRNA levels were examined to extrapolate further the relationship among cholesterol transport, cholesterol biosynthesis, and LHR mRNA expression. HCG injection caused a persistent increase in LDLR and HDLR mRNA levels, both of which remained elevated at 12 h. (Fig. 6, G–H). The time-courses of LDLR and HDLR mRNA expression were different from those of cholesterol biosynthesis enzymes (Fig. 6, D–F), which suggests that, although lipoprotein receptors, like the cholesterol biosynthesis enzymes, are up-regulated by hCG at the transcriptional level, the mechanisms of regulation are probably different.
Effect of hCG on ovarian sterol concentrations
It is known that the gene expression of cholesterol biosynthesis enzymes and plasma lipoprotein receptors is negatively regulated in cholesterol-rich environment, via a SREBP-dependent pathway (33, 34, 35). Thus, the effect of hCG administration on the cholesterol content in pseudopregnant rat ovaries was examined in relation to the coordinate up-regulation of Mvk and related proteins. The ovarian cholesterol levels were assayed at 2, 4, 12, 18, and 24 h, after hCG injection to d-5 pseudopregnant rats. The results presented in Fig. 7A show that, as expected, treatment with hCG caused a rapid decline in the total cellular cholesterol level, which reached its lowest level at 4 h and remained low even at 24 h, In contrast, the cholesterol content in control ovaries was unchanged over the period. Free cholesterol measurements followed a pattern similar to that seen with total cholesterol (Fig. 7B). These results indicate that, during the 24-h period after hCG down-regulation, the ovarian cholesterol content remains depleted, which explains the continuous high levels of LDLR and HDLR mRNAs.
FIG. 7. Regulation of total/free cholesterol levels by hCG in pseudopregnant rat ovary (0–24 h). Rats were injected with hCG or saline on the fifth day of pseudopregnancy, and ovaries were collected at 0, 2, 4, 12, 18, and 24 h. Data are expressed as the mean of three determinations ± SE. Statistical differences between treated and control samples are indicated by the symbol (*) (P < 0.05).
Discussion
Previous studies have shown that LHR mRNA down-regulation in response to a pharmacological dose of hCG is mediated by a specific LRBP (4, 7, 8). The identity of LRBP was established as Mvk, an enzyme involved in cholesterol biosynthesis (9). Furthermore, both ATP and mevalonate competed for the binding of Mvk to LHR mRNA, suggesting that the catalytic site of Mvk might participate in the binding of LHR mRNA either directly or indirectly (9). Structural studies of Mvk using bacterially expressed enzyme revealed its similarity to the GHMP family of proteins (36), which possess a ?--? loop known as ribosomal protein S5 domain 2-like fold similar to that found in proteins that interact with rRNA (9). These facts strongly support the notion that Mvk mediates LHR mRNA degradation by binding to the mRNA molecule specifically.
Although the presence of Mvk in the liver of rat and pig was demonstrated in the late 1950s (37), the cloning (10), purification (11), and structure/function relationship (38, 39, 40) of purified enzyme have been accomplished only more recently. Despite the extensive studies in the liver, only limited studies have been carried out in nonhepatic tissues (26). As a prelude to the study on the regulation of Mvk in ovary, we performed 3'RACE and determined that Mvk mRNA is present in a single molecular form in the ovary, which is identical with that found in the liver. Furthermore, Western blot analysis was conducted using an antibody against the N-terminal amino acids of Mvk (21). Though multiple immunoreactive bands were detected using ovarian samples (Fig. 4) as well as liver samples, the 42-kDa band appeared to be the predominant one, which is consistent with the report of Biardi et al. (21). In liver, Biardi et al. detected multiple bands using this antibody, but a single 42-kDa band was detected using antibodies against different epitopes of Mvk. Thus, we believe that there is only one 42-kDa Mvk protein in rat ovary, and other bands are artifacts.
After establishing that the mRNA transcript of Mvk in ovary is identical with that found in liver, the effects of hCG treatment on its expression and function were determined in the context of the down-regulation of LHR mRNA expression. Our results showed that hCG-induced down-regulation of LHR mRNA was demonstrable at 4 h after hormone treatment, and this loss of mRNA was coincident with an increase in LHR mRNA binding activity of Mvk from the S-100 fraction. Western blot results also showed a corresponding increase, suggesting that the increased LHR mRNA binding is a result of elevated Mvk protein content in the cytosolic fractions of the ovarian cells. This elevation was preceded by a short-term increase of Mvk mRNA levels, as demonstrated by both Northern blot (Fig. 3) and real-time PCR data (Fig. 6). The persistence of Mvk protein level beyond time periods when its mRNA level had returned to the control level suggests that the decay rate of protein is slower than that of mRNA. An alternate explanation is that hCG treatment may have triggered other events, such as increased translation or protein stabilization. Our data indicated that hCG-induced elevation of Mvk mRNA levels lasted for a short duration, starting immediately after hCG injection and ending within a few hours. In fact, after 12 h, Mvk mRNA expression levels showed an increase in both hCG-treated and control rat ovaries. Thus, the onset of the loss of steady-state levels of LHR mRNA appears to closely correlate with the early effect of hCG treatment on Mvk expression.
In the ovary, cholesterol required for steroidogenesis and other cellular functions is acquired through de novo synthesis and through receptor-mediated uptake from LDL and HDL (30, 41, 42). The receptors for LDL and HDL as well as HMGR in ovary have been shown to be induced by LH/hCG (30, 43, 44). Because cholesterol transport/synthesis and steroidogenesis in the ovary is intimately associated with LHR expression, the involvement of Mvk as a protein that regulates LHR mRNA expression is highly significant. Our results, using real-time PCR, demonstrate that hCG not only stimulated the expression of Mvk leading to accelerated LHR mRNA decay but also increased the expression of a number of sterol-responsive genes that contribute to cholesterol accumulation in the ovary (Fig. 8). So far, this is the first report showing that, in pseudopregnant rat ovary, expression of cholesterol biosynthesis enzymes and lipoprotein receptors are coordinately up-regulated by hCG. Increased expression of ovarian Mvk, together with other enzymes such as HMGS and HMGR, is expected to contribute to cellular cholesterol accumulation, although it is presently unclear as to how Mvk serves its dual functions, as an enzyme and an RNA-binding protein. Whether there is a factor that is required for switching Mvk’s function from a catalytic protein to an RNA-binding protein remains an open question at this time.
FIG. 8. Proposed model of LHR down-regulation by LH/hCG in luteinized ovarian cells. LH/hCG binding to the LHR activates the cAMP signal cascade and increases the transcription of a series of cholesterol biosynthesis enzymes and steroidogenesis. An increase in Mvk protein level was seen 4 h after hCG injection. This leads to an increase in Mvk-LHR mRNA complex formation and accelerates LHR mRNA decay. The decrease of LHR mRNA level culminates in the loss of cell surface LHR and produces a transient abolishment of the signal cascade. PP, Pyrophosphate.
It should be noticed that, whereas the expression of cholesterol biosynthesis enzymes returned to control level within a few hours, the expression of LDLR and HDLR mRNA remains high up to 12 h after hCG treatment. It is interesting to note that the cellular cholesterol levels remain low in hCG-treated animals compared with the control (Fig. 7). This may point out the different mechanisms by which plasma lipoprotein receptors and cholesterol biosynthesis enzymes are regulated. Lopez et al. (17) reported that, in PMSG-primed rats, hCG treatment induces cholesterol depletion, leading to increased production of sterol regulatory element-binding protein-1a (SREBP-1a). This factor and SF-1, a transcription factor activated directly by cAMP, synergize the induction of HDLR expression (17). Our present results, that the expression of HDL and LDR remained high at a time when cell surface LHR was completely down-regulated, are consistent with this notion. The rapid depletion of ovarian cholesterol after hCG injection suggests that cellular cholesterol status may be a factor that plays a role in increasing Mvk expression. However, the rapid decrease of Mvk mRNA levels between 4–6 h, despite the cholesterol depletion prevailing for more than 24 h, cannot explain this simple relationship. Although we have not examined the direct transcriptional regulation of Mvk by cAMP, available evidence (45) points to such a mechanism.
In summary, we present evidence for a novel mechanism for the regulation of LHR mRNA expression in the ovary. Our results show that hCG stimulates Mvk expression, which leads to increased binding to LHR mRNA and accelerates its decay. Furthermore, hCG coordinately up-regulates the expression of Mvk, along with other cholesterol biosynthesis enzymes and lipoprotein receptors. (Fig. 8).
Acknowledgments
The authors express their appreciation to Dr. Anil K Nair, Christine Clouser, Helle Peegel, Dr. Roberto Towns, and Dr. Pradeep Kayampilly for critical reading of the manuscript.
References
Ascoli M, Fanelli F, Segaloff DL 2002 The lutropin/choriogonadotropin receptor, a 2002 perspective. Endocr Rev 23:141–174
Menon KMJ, Munshi UM, Clouser CL, Nair AK 2004 Regulation of luteinizing hormone/human chorionic gonadotropin receptor expression: a perspective. Biol Reprod 70:861–866
Hoffman YM, Peegel H, Sprock MJ, Zhang QY, Menon KMJ 1991 Evidence that human chorionic gonadotropin/luteinizing hormone receptor down-regulation involves decreased levels of receptor messenger ribonucleic acid. Endocrinology 128:388–393
Kash JC, Menon KMJ 1998 Identification of a hormonally regulated luteinizing hormone/human chorionic gonadotropin receptor mRNA binding protein. Increased mRNA binding during receptor down-regulation. J Biol Chem 273:10658–10664
Lu DL, Peegel H, Mosier SM, Menon KMJ 1993 Loss of lutropin/human choriogonadotropin receptor messenger ribonucleic acid during ligand-induced down-regulation occurs post transcriptionally. Endocrinology 132:235–240
LaPolt PS, Oikawa M, Jia XC, Dargan C, Hsueh AJ 1990 Gonadotropin-induced up- and down-regulation of rat ovarian LH receptor message levels during follicular growth, ovulation and luteinization. Endocrinology 126:3277–3279
Kash JC, Menon KMJ 1999 Sequence-specific binding of a hormonally regulated mRNA binding protein to cytidine-rich sequences in the lutropin receptor open reading frame. Biochemistry 38:16889–16897
Nair AK, Kash JC, Peegel H, Menon KMJ 2002 Post-transcriptional regulation of luteinizing hormone receptor mRNA in the ovary by a novel mRNA-binding protein. J Biol Chem 277:21468–21473
Nair AK, Menon KMJ 2004 Isolation and characterization of a novel trans-factor for luteinizing hormone receptor mRNA from ovary. J Biol Chem 279:14937–14944
Tanaka RD, Lee LY, Schafer BL, Kratunis VJ, Mohler WA, Robinson GW, Mosley ST 1990 Molecular cloning of mevalonate kinase and regulation of its mRNA levels in rat liver. Proc Natl Acad Sci USA 87:2872–2876
Tanaka RD, Schafer BL, Lee LY, Freudenberger JS, Mosley ST 1990 Purification and regulation of mevalonate kinase from rat liver. J Biol Chem 265:2391–2398
Gwynne JT, Strauss III JF 1982 The role of lipoproteins in steroidogenesis and cholesterol metabolism in steroidogenic glands. Endocr Rev 3:299–329
Azhar S, Reaven E 2002 Scavenger receptor class BI and selective cholesteryl ester uptake: partners in the regulation of steroidogenesis. Mol Cell Endocrinol 195:1–26
Li X, Peegel H, Menon KMJ 1998 In situ hybridization of high density lipoprotein (scavenger, type 1) receptor messenger ribonucleic acid (mRNA) during folliculogenesis and luteinization: evidence for mRNA expression and induction by human chorionic gonadotropin specifically in cell types that use cholesterol for steroidogenesis. Endocrinology 139:3043–3049
Hwang J, Menon KMJ 1983 Characterization of low density and high density lipoprotein receptors in the rat corpus luteum and regulation by gonadotropin. J Biol Chem 258:8020–8027
Bishop RW, Chambliss KL, Hoffmann GF, Tanaka RD, Gibson KM 1998 Characterization of the mevalonate kinase 5'-untranslated region provides evidence for coordinate regulation of cholesterol biosynthesis. Biochem Biophys Res Commun 242:518–524
Lopez D, McLean MP 1999 Sterol regulatory element-binding protein-1a binds to cis elements in the promoter of the rat high density lipoprotein receptor SR-BI gene. Endocrinology 140:5669–5681
Edwards PA, Ericsson J 1998 Signaling molecules derived from the cholesterol biosynthetic pathway: mechanisms of action and possible roles in human disease. Curr Opin Lipidol 9:433–440
Chomczynski P, Mackey K 1995 Short technical reports. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. Biotechniques 19:942–945
Wang L, Xie LP, Huang WQ, Yao B, Pu RL, Zhang RQ 2001 Presence of gonadotropin-releasing hormone (GnRH) and its mRNA in rat pancreas. Mol Cell Endocrinol 172:185–191
Biardi L, Sreedhar A, Zokaei A, Vartak NB, Bozeat RL, Shackelford JE, Keller GA, Krisans SK 1994 Mevalonate kinase is predominantly localized in peroxisomes and is defective in patients with peroxisome deficiency disorders. J Biol Chem 269:1197–1205
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F 2002 Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034
Chan YL, Paz V, Olvera J, Wool IG 1990 The primary structure of rat ribosomal protein S16. FEBS Lett 263:85–88
Bligh EG, Dyer WJ 1959 A rapid method of total lipid extraction and purification. Can J Med Sci 37:911–917
Li X, Peegel H, Menon KMJ 2001 Regulation of high density lipoprotein receptor messenger ribonucleic acid expression and cholesterol transport in theca-interstitial cells by insulin and human chorionic gonadotropin. Endocrinology 142:174–181
Flint AP 1970 The activity and kinetic properties of mevalonate kinase in superovulated rat ovary. Biochem J 120:145–150
Leo CP, Pisarska MD, Hsueh AJ 2001 DNA array analysis of changes in preovulatory gene expression in the rat ovary. Biol Reprod 65:269–276
Bowen JM, Telleria CM, Towns R, Keyes PL 2000 Down-regulation of long-form prolactin receptor mRNA during prolactin-induced luteal regression. Eur J Endocrinol 143:285–292
Dasmahapatra AK, Trewin AL, Hutz RJ 2002 Estrous cycle-regulated expression of CYP1B1 mRNA in the rat ovary. Comp Biochem Physiol B Biochem Mol Biol 133:127–134
Golos TG, Strauss III JF 1987 Regulation of low density lipoprotein receptor gene expression in cultured human granulosa cells: roles of human chorionic gonadotropin, 8-bromo-3',5'-cyclic adenosine monophosphate, and protein synthesis. Mol Endocrinol 1:321–326
Wood JR, Strauss III JF 2002 Multiple signal transduction pathways regulate ovarian steroidogenesis. Rev Endocr Metab Disord 3:33–46
Horton JD, Goldstein JL, Brown MS 2002 SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 109:1125–1131[Free Full Text]
Goldstein JL, Brown MS 1990 Regulation of the mevalonate pathway. Nature 343:425–430
Brown MS, Goldstein JL 1999 A proteolytic pathway that control the cholesterol content of membranes, cells, and blood. Proc Natl Acad Sci USA 96:11041–11048
Brown MS, Goldstein JL 1997 The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89:331–340
Krepkiy D, Miziorko HM 2004 Identification of active site residues in mevalonate diphosphate decarboxylase: implications for a family of phosphotransferases. Protein Sci 13:1875–1881
Levy HR, Popjak G 1960 Studies on the biosynthesis of cholesterol. 10. Mevalonic kinase from liver. Biochem J 75:417–428
Potter D, Wojnar JM, Narasimhan C, Miziorko HM 1997 Identification and functional characterization of an active-site lysine in mevalonate kinase. J Biol Chem 272:5741–5746
Potter D, Miziorko HM 1997 Identification of catalytic residues in human mevalonate kinase. J Biol Chem 272:25449–25454
Fu Z, Wang M, Potter D, Miziorko HM, Kim JJ 2002 The structure of a binary complex between a mammalian mevalonate kinase and ATP: insights into the reaction mechanism and human inherited disease. J Biol Chem 277:18134–18142
Rajendran KG, Hwang J, Menon KMJ 1983 Binding, degradation, and utilization of plasma high density and low density lipoproteins for progesterone production in cultured rat luteal cells. Endocrinology 112:1746–1753
Azhar S, Nomoto A, Leers-Sucheta S, Reaven E 1998 Simultaneous induction of an HDL receptor protein (SR-BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell model. J Lipid Res 39:1616–1628
Reaven E, Nomoto A, Leers-Sucheta S, Temel R, Williams DL, Azhar S 1998 Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian models. Endocrinology 139:2847–2856
Azhar S, Chen YD, Reaven GM 1984 Gonadotropin modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in desensitized luteinized rat ovary. Biochemistry 23:4533–4538
Medicherla S, Azhar S, Cooper A, Reaven E 1996 Regulation of cholesterol responsive genes in ovary cells: impact of cholesterol delivery systems. Biochemistry 35:6243–6250(Lei Wang and K. M. J. Men)