Association between LH receptor regulation and ovarian hyperstimulation syndrome in a rodent model.

Ovarian hyperstimulation syndrome (OHSS) is a common complication of ovarian stimulation associated with the administration of human chorionic gonadotropin (hCG) during assisted reproduction. We have determined the expression of luteinizing hormone receptor (Lhcgr) mRNA, vascular endothelial growth factor (VEGF), and its transcription factor, HIF1α, during the periovulatory period in a rodent model of OHSS and compared these results with normal ovulatory periods. These results showed that the downregulation of Lhcgr mRNA in response to conditions that mimic preovulatory LH surge was significantly impaired in the OHSS group compared to the complete downregulation seen in the control group. Most importantly, the downregulation of luteinizing hormone receptor mRNA expression following hCG administration was sustained in the control group up to 48 h, whereas it remained at significantly higher levels in the OHSS group. This impairment of hCG-induced Lhcgr downregulation in the OHSS group was accompanied by significantly elevated levels of VEGF and its transcription factor, HIF1α. Furthermore, the downregulation of Lhcgr that occurs in response to a preovulatory LH surge in normal cycles was accompanied by low levels of VEGF. This study shows that, while downregulation of Lhcgr as well as low VEGF levels are seen in response to a preovulatory LH surge in normal ovarian cycle, impaired Lhcgr downregulation and elevated VEGF levels were found in the OHSS group.

SREBP Plays a Regulatory Role in LH/hCG Receptor mRNA Expression in Human Granulosa-Lutein Cells.

CONTEXT: LH receptor (LHR) expression has been shown to be regulated posttranscriptionally by LHR mRNA binding protein (LRBP) in rodent and human ovaries. LRBP was characterized as mevalonate kinase. The gene that encodes mevalonate kinase is a member of a family of genes that encode enzymes involved in lipid synthesis and are regulated by the transcription factor sterol regulatory element binding proteins (SREBPs). OBJECTIVE: The current study examined the regulation of LHR mRNA expression in human granulosa-lutein cells in response to alterations in cholesterol metabolism. DESIGN: Using atorvastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase to inhibit cholesterol biosynthesis, we examined its effect on LHR mRNA expression. The effect of atorvastatin on SREBP and mRNA expression as well as LHR mRNA binding protein expression was examined. Finally, the effect of atorvastatin on human chorionic gonadotropin (hCG)-stimulated progesterone production and the expression of key steroidogenic enzymes was also examined. RESULTS: Statin treatment reduced LHR mRNA expression by increasing the levels of SREBP1a and SREBP2, leading to an increase in LRBP. RNA gel shift assay showed that increased binding of LHR mRNA to LRBP occurred in response to atorvastatin, leading to LHR mRNA degradation. The granulosa-lutein cells pretreated with atorvastatin also showed decreased responsiveness to hCG by decreasing the mRNA and protein expression of steroidogenic enzymes. Atorvastatin also attenuated LH/hCG-induced progesterone production. CONCLUSION: These results imply that LHR mRNA expression by the human granulosa-lutein cells is regulated by cholesterol, through a mechanism involving SREBP and SREBP cleavage activating protein serving as the cholesterol sensor.

miR-122 Regulates LHR Expression in Rat Granulosa Cells by Targeting Insig1 mRNA.

Luteinizing hormone/chorionic gonadotropin receptor (LHR) expression in the ovary is regulated by a messenger RNA (mRNA) binding protein, which specifically binds to the coding region of LHR mRNA. We have shown that miR-122, a short noncoding RNA, mediates LHR mRNA levels by modulating the expression of LHR mRNA-binding protein (LRBP) through the regulation of sterol regulatory element binding protein (SREBP) activation. The present results show that miR-122 regulates LRBP levels by increasing the processing of SREBP through the degradation of Insig1, the anchoring protein of SREBP. We present evidence showing that mRNA and protein levels of Insig1 undergo a time-dependent increase following the treatment of rat granulosa cells with follicle-stimulating hormone (FSH), which leads to a decrease in LRBP levels. Furthermore, overexpression of miR-122 using an adenoviral vector (AdmiR-122) abolished FSH-induced increases in Insig1 mRNA and protein. We further confirmed the role of Insig1 by showing that inhibition of Insig1 using a specific small interfering RNA prior to FSH treatment resulted in the abrogation of LHR upregulation. Silencing of Insig1 also reversed FSH-mediated decreases in SREBP and LRBP activation. These results show that decreased levels of miR-122 increase Insig1 and suppress SREBP processing in response to FSH stimulation of rat granulosa cells.

Regulation of Luteinizing Hormone Receptor mRNA Expression in the Ovary: The Role of miR-122.

The expression of luteinizing hormone receptor (LHR) in the mammalian ovary is regulated in response to changes in the secretion of follicle-stimulating hormone and luteinizing hormone by the anterior pituitary, at least in part, through posttranscriptional mechanisms. The steady-state levels of LHR mRNA are maintained by controlling its rate of degradation by an RNA-binding protein designated as LHR mRNA-binding protein (LRBP). LRBP forms a complex with LHR mRNA and targets it for degradation in the p bodies. miR-122, an 18 nucleotide noncoding RNA, regulates the expression of LRBP. Thus, the levels of miR-122 determine the cellular levels of LHR mRNA expression. This phenomenon has been examined during the induction of LHR mRNA expression that occurs during follicle maturation in response to rising levels of FSH. In this situation, miR-122 and LRBP levels decrease as LHR mRNA expression undergoes downregulation in response to preovulatory LH surge. miR-122 expression as well as LRBP levels show robust increases. The mechanism of induction of LRBP by miR-122 has also been discussed.

LHCGR Expression During Follicle Stimulating Hormone-Induced Follicle Growth Is Negatively Regulated by Eukaryotic Initiation Factor 5A.

We have shown that the transient changes in the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) messenger RNA (mRNA) during the ovarian cycle occurs, at least in part, through a posttranscriptional mechanism involving an LHCGR mRNA-binding protein (LRBP). Eukaryotic initiation factor 5A (eIF5A), an LRBP-interacting protein, participates in this process. eIF5A undergoes hypusination, a unique posttranslational modification that is necessary for its functions. This study examined the role of eIF5A in follicle-stimulating hormone (FSH)-induced LHCGR expression during follicular growth. Treatment of primary cultures of rat granulosa cells with FSH and 17ß-estradiol (E2) showed a time-dependent increase in LHCGR mRNA expression. Conversely, inhibition of endogenous hypusination of eIF5A using N1-guanyl-1,7-diaminoheptane (GC7), a hypusination inhibitor, showed a greater increase in LHCGR mRNA expression over that produced by FSH and E2 alone. Further studies were carried out to determine the mechanism by which inhibition of hypusination of eIF5A causes an increase in LHCGR mRNA expression. Because LHCGR expression is negatively regulated by LRBP, the effect of inhibiting hypusination of eIF5A on LRBP expression was examined. The results showed a decrease in the expression of LRBP mRNA and protein when hypusination of eIF5A was inhibited by GC7. Because LRBP promotes LHCGR mRNA degradation, the results of this study support the notion that by inhibiting eIF5A hypusination, FSH reduces the expression of LRBP. This increases LHCGR mRNA expression by abrogating the inhibitory action of LRBP.

Molecular regulation of LHCGR expression by miR-122 during follicle growth in the rat ovary.

We have previously reported that LHCGR expression in the ovary is regulated through a post-transcriptional mechanism involving an mRNA binding protein designated as LRBP, which is regulated, at least in part, by a non-coding RNA, miR-122. Our present study examined the regulatory role of miR-122 in FSH-induced LHCGR expression during follicle development. Treatment of rat granulosa cells concurrently with FSH and 17ß estradiol showed, as expected, a time-dependent increase in LHCGR mRNA levels as well as hCG-induced progesterone production. However, miR-122 expression was decreased during the early time periods, which preceded the increased expression of LHCGR mRNA. The role of miR-122 in FSH-induced LHCGR mRNA expression was then examined by overexpressing miR-122 prior to FSH stimulation by infecting granulosa cells with an adenoviral vector containing a miR-122 insert (AdmiR-122). Pretreatment with AdmiR-122 resulted in complete abrogation of FSH- mediated upregulation of LHCGR. AdmiR-122 also blocked FSH-induced decrease in LRBP expression and increased the binding of LHCGR mRNA to LRBP. Based on these results, we conclude that miR-122 plays a regulatory role in LHCGR expression by modulating LRBP levels during FSH-induced follicle growth.

miR-122 Regulates LH Receptor Expression by Activating Sterol Response Element Binding Protein in Rat Ovaries.

LH/human chorionic gonadotropin receptor (LHR) undergoes down-regulation during preovulatory LH surge or in response to exposure to a supraphysiological concentration of its ligands through a posttranscriptional mechanism involving an RNA binding protein designated as LHR mRNA binding protein (LRBP). miR-122, a short noncoding RNA, has been shown to mediate the up-regulation of LRBP. In the present study, we show that inhibition of miR-122 using a locked nucleic acid (LNA)-conjugated antagomir suppressed human chorionic gonadotropin (hCG)-induced up-regulation of LRBP as well as its association with LHR mRNA, as analyzed by RNA EMSA. Most importantly, inhibition of miR-122 resulted in the abolishment of hCG-mediated LHR mRNA down-regulation. We also show that the transcription factor, sterol regulatory element binding protein (SREBP) (SREBP-1a and SREBP-2 isoforms), is an intermediate in miR-122-mediated LHR mRNA regulation. HCG-stimulated increase in the activation of both SREBP-1a and SREBP-2 was inhibited by pretreatment with the miR-122 antagomir. The inhibition of cAMP/protein kinase A (PKA) and ERK pathways, upstream activators of miR-122, abolished SREBP activation after hCG treatment. SREBP-mediated regulation of LRBP expression is mediated by recruitment of LRBP promoter element to SREBP-1a, because chromatin immunoprecipitation assay revealed that association of LRBP promoter to SREBP was increased by hCG treatment. Pretreatment with miR-122 antagomir suppressed this response. Inhibition of SREBP activation by pretreating the rats with a chemical compound, fatostatin abrogated hCG-induced up-regulation of LRBP mRNA and protein. Fatostatin also inhibited LHR-LRBP mRNA-protein complex formation and LHR down-regulation. These results conclusively show that miR-122 plays a regulatory role in LH/hCG-induced LHR mRNA down-regulation by increasing LRBP expression through the activation of SREBP pathway.

Hypusination of eukaryotic initiation factor 5A via cAMP-PKA-ERK1/2 pathway is required for ligand-induced downregulation of LH receptor mRNA expression in the ovary.

Luteinizing hormone receptor (LHR) mRNA expression in the ovary is regulated post-transcriptionally by an LH receptor mRNA binding protein (LRBP). Eukaryotic initiation factor 5A (EIF5A), identified as an LRBP-interacting protein plays a crucial role in LHR mRNA expression. In this study, we have demonstrated that during hCG-induced LHR downregulation, a significant upregulation of eIF5A mRNA expression and hypusination of eIF5A protein occurs in a time dependent manner. Pretreatment with H89, a specific inhibitor of PKA, and U0126, a specific inhibitor of ERK1/2 significantly inhibited both hCG-induced eIF5A mRNA expression and hypusination of eIF5A protein. Pretreatment with GC7, a specific inhibitor of eIF5A hypusination significantly abolished hCG-induced LRBP mRNA and protein expression. Furthermore, GC7 pretreatment significantly inhibited hCG-induced interaction of LRBP with LHR mRNA as assessed by RNA electrophoretic mobility gel shift assay (REMSA). GC7 treatment also reversed LHR mRNA downregulation. Taken together, these results suggest that hCG-induced LHR mRNA downregulation is mediated by cAMP-PKA-ERK1/2 signaling leading to activation of eIF5A hypusination.

Eukaryotic initiation factor 5A plays an essential role in luteinizing hormone receptor regulation.

Down-regulation of LH receptor (LHR) in the ovary by its ligand is mediated by a specific RNA-binding protein, designated LH receptor mRNA-binding protein (LRBP), through translational suppression and mRNA degradation. Using yeast 2-hybrid screens, we previously identified eukaryotic initiation factor 5A (eIF5A) as one of the proteins that interacts with LRBP during LHR mRNA down-regulation. The present study examined the role of eIF5A and its hypusination in the context of LHR mRNA down-regulation. The association of eIF5A with LRBP or LHR mRNA was determined using immunoprecipitation and RNA immunoprecipitation assays. The results showed that the association of eIF5A with the LHR mRNA-LRBP complex increased significantly during down-regulation. Furthermore, gel fractionation and the hypusination activity assay both showed increased hypusination of eIF5A during LHR mRNA down-regulation. Abolishment of hypusination by pretreatment with the chemical inhibitor GC7 prevented the association of eIF5A with LHR mRNA and LRBP. Inhibition of hypusination also reduced the extent of ligand-induced down-regulation of LHR mRNA as well as the expression of functional LHRs assessed by real-time PCR and (125)I-human chorionic gonadotropin (hCG) binding assays, respectively. The loss of human chorionic gonadotropin-mediated downstream signaling during LHR down-regulation was also restored by inhibition of hypusination of eIF5A. Thus, the present study, for the first time, reveals the crucial role of eIF5A and its hypusination in the regulation of LHR expression in the ovary.

A novel small-molecule inhibitor of mcl-1 blocks pancreatic cancer growth in vitro and in vivo.

Using a high-throughput screening (HTS) approach, we have identified and validated several small-molecule Mcl-1 inhibitors (SMI). Here, we describe a novel selective Mcl-1 SMI inhibitor, 2 (UMI-77), developed by structure-based chemical modifications of the lead compound 1 (UMI-59). We have characterized the binding of UMI-77 to Mcl-1 by using complementary biochemical, biophysical, and computational methods and determined its antitumor activity against a panel of pancreatic cancer cells and an in vivo xenograft model. UMI-77 binds to the BH3-binding groove of Mcl-1 with Ki of 490 nmol/L, showing selectivity over other members of the antiapoptotic Bcl-2 family. UMI-77 inhibits cell growth and induces apoptosis in pancreatic cancer cells in a time- and dose-dependent manner, accompanied by cytochrome c release and caspase-3 activation. Coimmunoprecipitation experiments revealed that UMI-77 blocks the heterodimerization of Mcl-1/Bax and Mcl-1/Bak in cells, thus antagonizing the Mcl-1 function. The Bax/Bak-dependent induction of apoptosis was further confirmed using murine embryonic fibroblasts that are Bax- and Bak-deficient. In an in vivo BxPC-3 xenograft model, UMI-77 effectively inhibited tumor growth. Western blot analysis in tumor remnants revealed enhancement of proapoptotic markers and significant decrease of survivin. Collectively, these promising findings show the therapeutic potential of Mcl-1 inhibitors against pancreatic cancer and warrant further preclinical investigations.

Molecular interplay between cdk4 and p21 dictates G0/G1 cell cycle arrest in prostate cancer cells.

This study examined the effect of 3, 9-dihydroxy-2-prenylcoumestan (pso), a furanocoumarin, on PC-3 and C4-2B castration-resistant prostate cancer (CRPC) cell lines. Pso caused significant G0/G1 cell cycle arrest and inhibition of cell growth. Molecular analysis of cyclin (D1, D2, D3, and E), cyclin-dependent kinase (cdk) (cdks 2, 4, and 6), and cdk inhibitor (p21 and p27) expression suggested transcriptional regulation of the cdk inhibitors and more significant downregulation of cdk4 than of cyclins or other cdks. Overexpression of cdk4, or silencing of p21 or p27, overcame pso-induced G0/G1 arrest, suggesting that G0/G1 cell cycle arrest is a potential mechanism of growth inhibition in CRPC cells.

The role of Rab5a GTPase in endocytosis and post-endocytic trafficking of the hCG-human luteinizing hormone receptor complex.

This study examined the role of Rab5a GTPase in regulating hCG-induced internalization and trafficking of the hCG-LH receptor complex in transfected 293T cells. Coexpression of wild-type Rab5a (WT) or constitutively active Rab5a (Q79L) with LHR significantly increased hCG-induced LHR internalization. Conversely, coexpression of dominant negative Rab5a (S34N) with LHR reduced internalization. Confocal microscopy showed LHR colocalizing with Rab5a (WT) and Rab5a (Q79L) in punctuate structures. Coexpression of Rab5a (WT) and Rab5a (Q79L) with LHR significantly increased colocalization of LHR in early endosomes. Conversely, dominant negative Rab5a (S34N) decreased this colocalization. While Rab5a stimulated internalization of LHR, it significantly decreased LHR recycling to the cell surface and increased degradation. Dominant negative Rab5a (S34N) increased LHR recycling and decreased degradation. These results suggest that Rab5a plays a role in LHR trafficking by facilitating internalization and fusion to early endosomes, increasing the degradation of internalized receptor resulting in a reduction in LHR recycling.

Identification and characterization of proteins that selectively interact with the LHR mRNA binding protein (LRBP) in rat ovaries.

Luteinizing hormone receptor (LHR) mRNA binding protein (LRBP), identified as mevalonate kinase, has been shown to be a trans factor mediating the post-transcriptional regulation of LHR mRNA expression in ovaries. LRBP binds to the coding region of LHR mRNA and accelerates its degradation. Our previous studies in an in vitro system showed that LRBP represses the translation of LHR mRNA by forming an untranslatable ribonucleoprotein (mRNP) complex, further suggesting that the untranslatable mRNP complex is directed to the mRNA repression/decay machinery for subsequent mRNA turnover. In the present studies, we used yeast two-hybrid system to screen a cDNA library which was constructed from LHR down-regulated ovaries. Two proteins were identified interacting with LRBP: ribosomal protein S20 (RP S20) and ubiquitin conjugating enzyme 2i (UBCE2i). Their interactions with LRBP were confirmed by the mating assay, co-immunoprecipitation analyses and in vitro sumoylation assays. Furthermore, we show that LRBP is a target for modification by SUMO2/3 but not by SUMO1, at K256 and/or K345. Mutation of both lysine residues is sufficient to abrogate the sumoylation of LRBP. These findings suggest that the direct interaction of LRBP with the translation machinery, through RP S20, may be responsible for the transition of LHR mRNA to an untranslatable complex, and that sumoylation of LRBP may play a role in targeting the untranslatable mRNP complex to the mRNA decay machinery in specific cytoplasmic foci.