Evidence for the association of luteinizing hormone receptor mRNA-binding protein with the translating ribosomes during receptor downregulation.

Luteinizing hormone receptor (LHR) mRNA is post-transcriptionally regulated during ligand-induced downregulation. This process involves interaction of LHR mRNA with a specific mRNA-binding protein (LRBP), identified as mevalonate kinase (MVK), resulting in inhibition of translation followed by targeting the ribonucleoprotein complex to accelerated degradation. The present study investigated the endogenous association of LRBP with the translational machinery and its interaction with LHR mRNA during LH/hCG-induced downregulation. Ovaries were collected from rats that were injected with the ligand, hCG, to induce downregulation of LHR mRNA expression. Western blot analysis showed significantly higher levels of LRBP in polysomes from downregulated ovaries compared to controls. Western blot analysis of ribosome-rich fractions from FPLC-assisted gel filtration of post-mitochondrial supernatants confirmed the presence of LRBP in translating ribosomes isolated from the downregulated state but not from controls. The association of LRBP with LHR mRNA in the downregulated polysomes was demonstrated by immunoprecipitation with LRBP antibody followed by qPCR analysis of the associated RNA. Increased association of LHR mRNA with LRBP during downregulation was also demonstrated by subjecting the polysome-associated RNAs to oligo(dT) cellulose chromatography followed by immunoprecipitation and qPCR analysis. Additionally, analysis of in vitro translation of LHR mRNA showed increased inhibition of translation by polysomes from downregulated ovaries compared to control. This study provides strong in vivo and in vitro evidence to show that during ligand-induced downregulation, LRBP translocates to ribosomes and associates with LHR mRNA to form an untranslatable ribonucleoprotein complex and inhibits LHR mRNA translation, paving the way to its degradation.

The extracellular domain of luteinizing hormone receptor dictates its efficiency of maturation.

The processing of luteinizing hormone receptor (LHR) shows marked differences in different species. While the human LHR is predominantly expressed as the mature, 90kDa species, rat LHR exists mostly in the 70kDa precursor form. Since the extracellular domain of the LHR is unusually large in comparison with other G protein-coupled receptors, the present studies examined the role of extracellular domain in its processing. FLAG-tagged chimeric LH receptors were constructed by substituting the extracellular domain of the human receptor in rat LHR (hrr) and the extracellular domain of the rat receptor in human LHR (rhh). The intracellular processing, ligand binding and recycling of the chimeric receptors were compared with that of the wild type receptors in 293T cells. The results showed that the human and rat LHR were expressed predominantly as 90 and 70kDa species, respectively, as expected. The introduction of the rat extracellular domain into the human LHR (rhh) decreased the abundance of the mature form with an increase in the precursor form. Conversely, substitution of the extracellular domain of the rat LHR by the extracellular domain of the human LHR (hrr) led to an increase in the mature form with a corresponding decrease in the precursor form. Changes were also observed in the ligand binding and recycling of the wild type and chimeric receptors. These results suggest that the extracellular domain of the LHR is one of the determinants that confer its ability for proper maturation and cell surface expression.

Regulation of luteinizing hormone receptor mRNA expression by a specific RNA binding protein in the ovary.

The expression of LH receptor mRNA shows significant changes during different physiological states of the ovary. Previous studies from our laboratory have identified a post-transcriptional mechanism by which LH receptor mRNA is regulated following preovulatory LH surge or in response to hCG administration. A specific binding protein, identified as mevalonate kinase, binds to the open reading frame of LH receptor mRNA. The protein binding site is localized to nucleotides 203-220 of the LH receptor mRNA and exhibits a high degree of specificity. The expression levels of the protein show an inverse relationship to the LH receptor mRNA levels. The hCG-induced down-regulation of LH receptor mRNA can be mimicked by increasing the intracellular levels of cyclic AMP by a phosphodiesterase inhibitor. An in vitro mRNA decay assay showed that addition of the binding protein to the decay system caused accelerated LH receptor mRNA decay. Our results therefore show that LH receptor mRNA expression in the ovary is regulated post-transcriptionally by altering the rate of mRNA degradation by a specific mRNA binding protein.

The role of luteinizing hormone/human chorionic gonadotropin receptor-specific mRNA binding protein in regulating receptor expression in human ovarian granulosa cells.

CONTEXT: In normally cycling women, LH receptor mRNA expression undergoes transient down-regulation after the LH surge. The same phenomenon is also seen during a hormonally induced ovarian cycle where the LH receptor mRNA expression is down-regulated in response to the administration of human chorionic gonadotropin (hCG). Although the granulosa cells isolated from the follicular aspirates at this stage show a decline in the expression of LH receptor mRNA, this diminished receptor expression returns to control levels upon culturing in serum-containing medium. OBJECTIVE: To understand the mechanism of hCG-induced loss of LH receptor mRNA expression, a cytosolic fraction (S100) was isolated from the granulosa cells, and its ability to bind LH receptor mRNA was assayed by performing RNA electrophoretic mobility gel shift analysis. RESULTS: The results showed that the mRNA binding activity of the S100 fraction was induced in the freshly isolated granulosa cells (d 1) from the follicular aspirates collected from women who had been injected with hCG to induce ovulation. The LH receptor mRNA expression in granulosa cells on d 1, as assessed by real-time PCR and Northern blot analysis, was significantly suppressed. Both the expression of LH receptor mRNA and RNA binding activity in the S100 fraction were then assessed after culturing granulosa cells for 4 d. The results showed that the LH receptor mRNA expression was significantly higher on d 4 compared with that seen on d 1. However, the RNA binding activity of the S100 fraction was significantly decreased on d 4 compared with that seen on d 1. These results show an increased association of RNA binding protein during LH receptor mRNA down-regulation. CONCLUSION: The present results support the notion that LH receptor mRNA expression in the human ovaries is regulated by an RNA binding protein.

Evidence that palmitoylation of carboxyl terminus cysteine residues of the human luteinizing hormone receptor regulates postendocytic processing.

Palmitoylation is a well-conserved posttranslational modification among members of the G protein-coupled receptor superfamily. The present study examined the role of palmitoylation in endocytosis and postendocytic trafficking of the human LH receptor (LHR). Palmitoylation of the LHR was determined by incorporation of [3H]palmitic acid into wild-type (WT) or mutant receptor in which the potential palmitoylation sites, C643 and C644, were mutated to glycine residues. The WT receptor showed incorporation of [3H]palmitic acid into the mature 90-kDa form of the receptor whereas mutation of the two Cys residues abrogated this incorporation, indicating that Cys 643 and C644 are the sites of palmitoylation. The role of palmitoylation on endocytosis and postendocytic processing was examined by testing the ability of the WT and mutant receptor to undergo internalization, recycling, and lysosomal degradation. Compared with the WT receptor, the mutant receptor showed increased internalization and decreased recycling, suggesting that retention of palmitic acid residues at Cys 643 and 644 promotes LHR recycling. The role of palmitoylation on receptor recycling was substantiated by demonstrating that a different mutant, D578H LHR, which is deficient in palmitoylation, also recycled less efficiently. Furthermore, the data show that palmitoylation, not the rate of internalization, determines the efficiency of recycling. The present study shows that palmitoylation of cysteine residues 643 and 644 of the human LHR is a determinant of recycling.

Dihydrotestosterone inhibits granulosa cell proliferation by decreasing the cyclin D2 mRNA expression and cell cycle arrest at G1 phase.

Hyperandrogenism is known to perturb ovarian physiology resulting in anovulatory conditions. In the ovary, androgens produced by theca-interstitial cells are converted to estrogens in granulosa cells under the influence of FSH and LH. In some of the target organs, including the ovary, androgens are also converted into their 5alpha reduced metabolites. In the present study, we examined the molecular mechanism by which dihydrotestosterone (DHT), a 5alpha reduced metabolite of testosterone, mediates the inhibition of granulosa cell proliferation, using a rat model. Immature female rats were primed with estradiol, followed by DHT administration for 2 d and granulosa cells were cultured in the presence or absence of forskolin. Granulosa cells from the DHT-treated rats showed reduced [(3)H]thymidine incorporation into DNA and reduced cell number in response to forskolin stimulation, compared with control. The decreased responsiveness of DHT-treated granulosa cells to forskolin was not due to increased apoptosis because the expression of cleaved caspase 3 remained the same in both control and DHT-exposed granulosa cells stimulated with forskolin. Forskolin treatment stimulated the expression of cyclin D2 mRNA in control granulosa cells, whereas DHT treatment abolished this response. In vitro DHT treatment of granulosa cells for 48 h resulted in a cell cycle arrest with 70% of cells at G1 phase and 26% at S phase, and control cells exhibited a distribution of 42% and 55% at G1 and S phase, respectively. In conclusion, the present study shows that DHT inhibits the granulosa cell proliferation through a decrease in cyclin D2 mRNA expression, which leads to cell cycle arrest at the G1 phase.

Metformin: direct inhibition of rat ovarian theca-interstitial cell proliferation.

OBJECTIVE: To determine whether metformin has direct effects on ovarian theca-interstitial (T-I) cell proliferation through activation of adenosine monophosphate-activated protein kinase (AMPK). DESIGN: In vitro experimental study. SETTING: Academic medical center laboratory. ANIMAL(S): Immature Sprague-Dawley female rats. INTERVENTION(S): Ovarian T-I cells were isolated, purified, and cultured in the absence (control) or presence of insulin (1 µg/mL) with or without metformin or other activators/inhibitors of AMPK (AICAR, compound C). MAIN OUTCOME MEASURE(S): Proliferation assessed by determination of expression levels of proteins involved in cell cycle progression, cyclin D3, and cyclin-dependent kinase 4 (CDK4) with Western blot analysis, and determination of DNA synthesis with bromodeoxyuridine (BrdU) incorporation assay; activation of AMPK, Erk1/2, and S6K1 determined by Western blot analysis with the use of antibodies specific for the phosphorylated (activated) forms. RESULT(S): Metformin inhibited insulin-induced ovarian T-I cell proliferation and the up-regulation of the cell cycle regulatory proteins, cyclin D3 and CDK4. Metformin independently activated AMPK in a dose-dependent manner. Treatment with metformin inhibited insulin-induced activation of Erk1/2 and S6K1. This effect was reversed with the addition of compound C, a known AMPK inhibitor. CONCLUSION(S): Metformin directly inhibits proliferation of ovarian T-I cells via an AMPK-dependent mechanism. These findings further validate the potential benefits of metformin in the treatment of conditions associated with hyperinsulinemia and excessive growth of ovarian T-I cells (such as polycystic ovary syndrome).

Inhibitory effect of valproic acid on ovarian androgen biosynthesis in rat theca-interstitial cells.

The objective of the study was to evaluate the effect of valproic acid (VPA) on ovarian androgen biosynthesis in primary cultures of theca-interstitial (T-I) cells isolated from rat ovaries. Ovarian T-I cells were cultured with VPA in the presence or absence of hCG. VPA did not increase basal or hCG-stimulated androgen synthesis when added to primary cultures of T-I cells. However, the addition of VPA caused a marked concentration-dependent inhibitory effect on hCG-stimulated androstendione synthesis. Treatment of T-I cells with 8-Bromo-cAMP resulted in a marked increase in the production of androstenedione, and VPA inhibited this stimulatory effect, suggesting that the mechanism of VPA's inhibitory effect on androstenedione production occurs at a step after second messenger activation. Treatment of T-I cells with hCG resulted in a significant increase in the mRNA expression of steroidogenic enzymes CYP17A1 and 17ß-hydroxysteroid dehydrogenase. Addition of VPA sharply blunted the stimulatory effect of hCG, reducing the mRNA expression of the steroidogenic enzymes to basal levels. In conclusion, VPA exerts an inhibitory effect on hCG-stimulated androgen synthesis in rat T-I cells.

LH/hCG-stimulated androgen production and selective HDL-cholesterol transport are inhibited by a dominant-negative CREB construct in primary cultures of rat theca-interstitial cells.

Theca-interstitial (T-I) cells synthesize androgens that are converted to estrogen by the granulosa cells. In rat ovary, T-I cells primarily utilize HDL-derived cholesteryl esters (CE) as a precursor for androgen synthesis. The HDL-CE is delivered to steroidogenic cells by a process termed "selective" uptake in which CE is internalized without the simultaneous uptake of apolipoprotein(s). This process is mediated by an HDL receptor, scavenger receptor class B, type I (SR-BI) and is stimulated by trophic hormone (LH/hCG), which also activates the cAMP cascade. In this study, we tested whether the adenoviral (Ad)-mediated introduction of a dominant-negative analog of cyclic AMP response element binding protein (A-CREB) inhibits the stimulatory effect of LH/hCG on the selective uptake of high-density lipoprotein (HDL)-cholesterol and androgen production in primary cultures of rat T-I cells. Androstenedione production by cultured T-I cells was stimulated by hCG and by the adenoviral overexpression of wtCREB. Additionally, the stimulatory effect observed with hCG was amplified in the presence of HDL. Androgen synthesis was increased 17-fold in the presence of HDL and hCG but the stimulatory effect of hCG was inhibited by Ad A-CREB by approx 70%. In the selective up-take studies, cell-surface association of the labeled HDL was significantly enhanced by hCG treatment, and this effect was inhibited by Ad A-CREB. The selective uptake of HDL-cholesterol was also enhanced by hCG but exposure to Ad A-CREB also abrogated this effect. It is concluded that CREB plays an intermediary role in the stimulatory action of LH/hCG on androgen synthesis and the selective uptake of HDL-cholesterol in T-I cells.

Post-transcriptional regulation of luteinizing hormone receptor mRNA in the ovary by a novel mRNA-binding protein.

Luteinizing hormone (LH) receptor mRNA is post-transcriptionally regulated. An ovarian cytosolic LH receptor mRNA-binding protein (LRBP) identified in our laboratory binds to a polypyrimidine-rich bipartite sequence in the coding region of LH receptor mRNA. The present studies show a role for LRBP in the regulation of LH receptor mRNA. We demonstrated that increased LH receptor mRNA degradation occurs during hormone-induced LH receptor down-regulation. Furthermore, increased degradation of LH receptor mRNA was seen when partially purified LRBP was included in an in vitro mRNA decay reaction. The LH receptor mRNA binding activity of LRBP measured by RNA electrophoretic mobility shift analysis showed an inverse relationship to LH receptor mRNA levels during different physiological states. These results suggest that LRBP is a physiological regulator of LHR mRNA expression in the ovary and provides a novel mechanism for the regulation of LH receptor expression in the ovary.