K. M. J. Menon

Regulation of Luteinizing Hormone/Human Chorionic Gonadotropin Receptor Expression: A Perspective

Abstract The LH/hCG receptor, a member of the G protein coupled receptor family mediates the cellular actions of LH in the ovary. A considerable amount of information regarding its structure, mechanism of activation, and regulation of expression has emerged in recent years. Here we provide a brief overview of the current information on the structural organization of the receptor and the mechanism of receptor mediated signaling as well as an in-depth discussion on recent developments pertaining to the regulation of receptor expression. Specifically, we describe studies from our laboratory showing that the posttranscriptional regulation of the receptor involves an LH/hCG receptor mRNA-binding protein. We also propose a model to explain the loss of steady-state LH/hCG receptor mRNA levels during receptor down-regulation.

Structure, Function and Regulation of Gonadotropin Receptors- A Perspective

Luteinizing hormone receptor and follicle stimulating hormone receptor play a crucial role in female and male reproduction. Significant new information has emerged about the structure, mechanism of activation, and regulation of expression of these receptors. Here we provide an overview of the current information on those aspects with an in-depth discussion of the recent developments in the post-transcriptional mechanism of LH receptor expression mediated by a specific LH receptor mRNA binding protein, designated as LRBP. LRBP was identified by electrophoretic gel mobility shift assay using cytosolic fractions from ovaries in the down regulated state. LRBP was purified, its binding site on LH receptor mRNA was identified and characterized. During ligand-induced down regulation, LRBP expression is increased through the cAMP/PKA and ERK signaling pathway, is translocated to translating ribosomes, binds LH receptor mRNA and forms an untranslatable ribonucleoprotein complex. This complex is then routed to the mRNA degradation machinery resulting in diminished levels of both LHR mRNA and cell surface expression of LH receptor. The studies leading to these conclusions are presented.

Follicle-Stimulating Hormone Inhibits Adenosine 5′-Monophosphate-Activated Protein Kinase Activation and Promotes Cell Proliferation of Primary Granulosa Cells in Culture through an Akt-Dependent Pathway

FSH, acting through multiple signaling pathways, regulates the proliferation and growth of granulosa cells, which are critical for ovulation. The present study investigated whether AMP-activated protein kinase (AMPK), which controls the energy balance of the cell, plays a role in FSH-mediated increase in granulosa cell proliferation. Cells isolated from immature rat ovaries were grown in serum-free, phenol red free DMEM-F12 and were treated with FSH (50 ng/ml) for 0, 5, and 15 min. Western blot analysis showed a significant reduction in AMPK activation as observed by a reduction of phosphorylation at thr 172 in response to FSH treatment at all time points tested. FSH also reduced AMPK phosphorylation in a dose-dependent manner with maximum inhibition at 100 ng/ml. The chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, 0.5 mm) increased the cell cycle inhibitor p27 kip expression significantly, whereas the AMPK inhibitor (compound C, 20 microm) and FSH reduced p27kip expression significantly compared with control. FSH treatment resulted in an increase in the phosphorylation of AMPK at ser 485/491 and a reduction in thr 172 phosphorylation. Inhibition of Akt phosphorylation using Akt inhibitor VIII reversed the inhibitory effect of FSH on thr 172 phosphorylation of AMPK, whereas ERK inhibitor U0126 had no effect. These results show that FSH, through an Akt-dependent pathway, phosphorylates AMPK at ser 481/495 and inhibits its activation by reducing thr 172 phosphorylation. AMPK activation by 5-amino-imidazole-4-carboxamide-1-beta-D-ribofuranoside treatment resulted in a reduction of cell cycle regulatory protein cyclin D2 mRNA expression, whereas FSH increased the expression by 2-fold. These results suggest that FSH promotes granulosa cell proliferation by increasing cyclin D2 mRNA expression and by reducing p27 kip expression by inhibiting AMPK activation through an Akt-dependent pathway.

Regulation of high density lipoprotein receptor messenger ribonucleic acid expression and cholesterol transport in theca-interstitial cells by insulin and human chorionic gonadotropin

The synthesis of androgens by theca-interstitial cells is stimulated by LH and the insulin/insulin-like growth factor I (IGF-I) system. An essential element of the steroidogenesis is the uptake of plasma cholesterol and transformation to steroid hormones. In the rat, the uptake of cholesterol by the theca-interstitial cells is mediated by the high density lipoprotein receptor. The goal of the present study was to examine whether insulin has any effect on cholesterol delivery into theca-interstitial cells. The effects of insulin and hCG on the expression of the high density lipoprotein receptor (SR-BI) messenger RNA (mRNA) and intracellular cholesterol levels were examined in rat theca-interstitial cells under in vivo and in vitro conditions. Twenty-five-day-old rats were treated with insulin, hCG, or insulin followed by hCG. The expression of SR-BI mRNA was then examined in ovaries enriched in theca-interstitial cell population by Northern blot analysis. Treatment with insulin increased the expression of ...

The effect of insulin on aromatase activity in isolated human endometrial glands and stroma

Hyperinsulinemic states have been associated with an increased incidence of estrogen-dependent endometrial neoplasia. To study the effect of insulin on the ability of endometrium to aromatize androgens to estrogens, late proliferative endometrium was obtained from normally cycling women at the time of indicated surgery, separated into component glands and stroma, and grown to confluence. Separated gland and stromal cultures were incubated in triplicate with increasing insulin concentrations and epidermal growth factor. Aromatase activity was assayed by the production of tritiated water from tritium-labeled androstenedione. The activity was noted to increase proportionally with increasing concentrations of insulin greater than 10 U/ml, and the effect was specific. These data suggest the following conclusions: (1) Insulin stimulates aromatase activity in both endometrial glands and stroma; (2) hyperinsulinemia may predispose to endometrial neoplasia by enhancing endogenous endometrial estrogen production.

Human Chorionic Gonadotropin Stimulates Theca-Interstitial Cell Proliferation and Cell Cycle Regulatory Proteins by a cAMP-Dependent Activation of AKT/mTORC1 Signaling Pathway

In addition to playing a cardinal role in androgen production, LH also regulates growth and proliferation of theca-interstitial (T-I) cells. Here, we show for the first time that LH/human chorionic gonadotropin (hCG) regulates T-I cell proliferation via the mammalian target of rapamycin complex 1 (mTORC1) signaling network. LH/hCG treatment showed a time-dependent stimulation of T-I cell proliferation and phosphorylation of protein kinase B (AKT), ERK1/2, and ribosomal protein (rp)S6 kinase 1 (S6K1), and its downstream effector, rpS6. Pharmacological inhibition of ERK1/2 signaling did not block the hCG-induced phosphorylation of tuberin, the upstream regulator of mTORC1 or S6K1, the downstream target of mTORC1. However, inhibition of AKT signaling completely blocked the hCG response. Furthermore, the AKT-specific inhibitor abolished forskolin (FSK)-stimulated phosphorylation of AKT, tuberin, S6K1, and rpS6. Human CG and FSK-mediated phosphorylation of AKT and downstream targets of mTORC1 were attenuated by inhibition of adenylyl cyclase. Pharmacologic targeting of mTORC1 with rapamycin also abrogated hCG or FSK-induced phosphorylation of S6K1, rpS6, and eukaryotic initiation factor 4E binding protein 1. In addition, hCG or FSK-mediated up-regulation of the cell cycle regulatory proteins cyclin-dependent kinase 4, cyclin D3, and proliferating cell nuclear antigen was blocked by rapamycin. These results were further confirmed by demonstrating that knockdown of mTORC1 using small interfering RNA abolished hCG-mediated increases in cell proliferation and the expression of cyclin D3 and proliferating cell nuclear antigen. Taken together, the present studies show a novel intracellular signaling pathway for T-I cell proliferation involving LH/hCG-mediated activation of the AKT/mTORC1 signaling cascade.

A novel post-transcriptional mechanism of regulation of luteinizing hormone receptor expression by an RNA binding protein from the ovary

Luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor, a member of the rhodopsin/beta(2) adrenergic receptor subfamily of G-protein coupled receptors, is expressed primarily in the gonads and essential for the regulation of reproductive function. In the ovary, the expression of the receptor is post-transcriptionally regulated under physiological conditions. Studies from our laboratory showed that the ligand-induced down-regulation of the receptor occurs by accelerated degradation of the mRNA rather than by decreased transcription. We have identified a cytoplasmic LHR mRNA binding protein (LRBP) as a trans-acting factor in regulating LHR mRNA levels. LRBP binds to the coding region of LHR mRNA and causes accelerated degradation of mRNA. The RNA binding activity of LRBP was found to be inversely correlated to LH/hCG receptor mRNA levels. LRBP was purified to homogeneity and its identity was established as mevalonate kinase by N-terminal microsequencing and MALDI analysis. Mevalonate kinase, an enzyme involved in de novo synthesis of cholesterol, belongs to the GHMP family of kinases having a potential RNA binding fold. The expression of MVK mRNA and MVK protein levels were induced in response to hCG treatment prior to the down-regulation of LH/hCG receptor mRNA expression. A model for the post-transcriptional regulation of LH/hCG receptor in the ovary by mevalonate kinase is proposed.

Luteinizing hormone receptor mRNA down-regulation is mediated through ERK-dependent induction of RNA binding protein.

The ligand-induced down-regulation of LH receptor (LHR) expression in the ovaries, at least in part, is regulated by a posttranscriptional process mediated by a specific LH receptor mRNA binding protein (LRBP). The LH-mediated signaling pathways involved in this process were examined in primary cultures of human granulosa cells. Treatment with 10 IU human chorionic gonadotropin (hCG) for 12 h resulted in the down-regulation of LHR mRNA expression while producing an increase in LHR mRNA binding to LRBP as well as a 2-fold increase in LRBP levels. The activation of ERK1/2 pathway in LH-mediated LHR mRNA down-regulation was also established by demonstrating the translocation of ERK1/2 from the cytosol to the nucleus using confocal microcopy. Inhibition of protein kinase A using H-89 or ERK1/2 by U0126 abolished the LH-induced LHR mRNA down-regulation. These treatments also abrogated both the increases in LRBP levels as well as the LHR mRNA binding activity. The abolishment of the hCG-induced increase in LRBP levels and LHR mRNA binding activity was further confirmed by transfecting granulosa cells with ERK1/2 specific small interfering RNA. This treatment also reversed the hCG-induced down-regulation of LHR mRNA. These data show that LH-regulated ERK1/2 signaling is required for the LRBP-mediated down-regulation of LHR mRNA.

A novel mechanism for the modulation of luteinizing hormone receptor mRNA expression in the rat ovary.

Luteinizing hormone receptor (LHR) is a G-protein-coupled receptor that exerts its effects mainly through increased cAMP synthesis. Our previous studies have shown that a ovarian cytosolic protein, designated as LHR mRNA binding protein (LRBP) is an important regulator of the steady state levels of LHR expression. To test whether LHR mRNA expression is modulated by cAMP through LRBP activity, we used rolipram, a type IV phosphodiesterase inhibitor that is known to promote intracellular cAMP accumulation. On day 4 of pseudopregnancy, rats were treated with rolipram (1.25 mg/injection) to raise intracellular levels of cAMP. In order to maintain higher cAMP levels, up to four injections of rolipram were given, with the last injection 4 h before collecting the ovaries. Measurement of cAMP levels showed an increase (p< or =0.05) at 8, 12, and 24 h after rolipram injections at total dosages of 2.5, 3.75 and 5.0 mg/rat, respectively. Northern blot analysis of LHR mRNA showed that rolipram treatment also markedly reduced ovarian LHR mRNA levels by up to 75%. LHR mRNA binding activity of LRBP, assayed by RNA electrophoretic mobility shift analysis, using S-100 fractions from control or rolipram-treated ovaries showed increased LHR mRNA binding activity in the S-100 fractions from rolipram treated groups. These data indicate that chronic elevation of ovarian cAMP leads to a decreased expression of LHR mRNA with a concomitant increase in LHR mRNA binding activity of LRBP.

Role of palmitoylation of conserved cysteine residues of luteinizing hormone/human choriogonadotropin receptors in receptor down-regulation.

The conserved cysteine residues 621 and 622 of luteinizing hormone/human chorionic gonadotropin receptors were converted to serine (C621S, C622S, C621/622S) and glycine residues (C621/C622G) by site directed mutagenesis. The wild type and mutant receptor cDNAs were cloned into the mammalian expression vector (PCMV4) and human embryonic kidney cells (293 cells) were transiently transfected with these constructs. Equilibrium binding studies with [(125)I]hCG (human chorionic gonadotropin) showed that the mutant and wild type receptors expressed on the cell surface exhibited similar K(d). The effect of mutation of the conserved cysteine residues on the ability of the receptors to undergo ligand-induced down-regulation was then tested. In vitro exposure of cells expressing the wild type receptor to a saturating concentration of human chorionic gonadotropin (100 ng/ml) for 24 h resulted in modest down-regulation of receptors. The palmitoylation deficient mutants, C621S, C622S, C621/622S and C621/622G, showed increased down-regulation compared with the wild type receptor. The extent of down-regulation of the mutant receptors correlated with increased internalization of the receptor. Additionally, the G protein coupling efficiency of the palmitoylation deficient mutants was not different from the wild type since the EC(50)s for cyclic AMP (cAMP) production were identical in both groups. These studies demonstrate that palmitoylation deficient mutants are more prone to ligand-induced receptor down-regulation. Furthermore, abrogation of palmitoylation by mutagenesis showed no effect on the efficiency of the palmitoylation deficient mutants to couple to Gs protein.