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.

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.

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.

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.