Song Ling Poon

Leptin interferes with 3',5'-Cyclic Adenosine Monophosphate (cAMP) signaling to inhibit steroidogenesis in human granulosa cells

BackgroundObesity has been linked to an increased risk of female infertility. Leptin, an adipocytokine which is elevated during obesity, may influence gonadal function through modulating steroidogenesis in granulosa cells.MethodsThe effect of leptin on progesterone production in simian virus 40 immortalized granulosa (SVOG) cells was examined by Enzyme linked immunosorbent assay (ELISA). The effect of leptin on the expression of the steroidogenic enzymes (StAR, P450scc, 3betaHSD) in SVOG cells was examined by real-time PCR and Western blotting. The mRNA expression of leptin receptor isoforms in SVOG cells were examined by using PCR. SVOG cells were co-treated with leptin and specific pharmacological inhibitors to identify the signaling pathways involved in leptin-reduced progesterone production. Silencing RNA against leptin receptor was used to determine that the inhibition of leptin on cAMP-induced steroidogenesis acts in a leptin receptor-dependent manner.Results and ConclusionIn the present study, we investigated the cellular mechanisms underlying leptin-regulated steroidogenesis in human granulosa cells. We show that leptin inhibits 8-bromo cAMP-stimulated progesterone production in a concentration-dependent manner. Furthermore, we show that leptin inhibits expression of the cAMP-stimulated steroidogenic acute regulatory (StAR) protein, the rate limiting de novo protein in progesterone synthesis. Leptin induces the activation of ERK1/2, p38 and JNK but only the ERK1/2 (PD98059) and p38 (SB203580) inhibitors attenuate the leptin-induced inhibition of cAMP-stimulated StAR protein expression and progesterone production. These data suggest that the leptin-induced MAPK signal transduction pathway interferes with cAMP/PKA-stimulated steroidogenesis in human granulosa cells. Moreover, siRNA mediated knock-down of the endogenous leptin receptor attenuates the effect of leptin on cAMP-induced StAR protein expression and progesterone production, suggesting that the effect of leptin on steroidogenesis in granulosa cells is receptor dependent. In summary, leptin acts through the MAPK pathway to downregulate cAMP-induced StAR protein expression and progesterone production in immortalized human granulosa cells. These results suggest a possible mechanism by which gonadal steroidogenesis could be suppressed in obese women.

The in Vivo and in Vitro Stimulatory Effects of Cordycepin on Mouse Leydig Cell Steroidogenesis

Cordycepin, a pure compound of Cordyceps sinensis (CS), is known as an adenosine analog. We have found that CS stimulated Leydig cell steroidogenesis. Here we investigated the in vivo and in vitro effects of cordycepin in primary mouse Leydig cell steroidogenesis. The results indicate that cordycepin increased the plasma testosterone concentration. Cordycepin also stimulated in vitro mouse Leydig cell testosterone production in dose- and time-dependent manners. We further observed that cordycepin regulated the mRNA expression of the A1, A2a, A2b, and A3 adenosine receptors in the mouse Leydig cells, and that antagonists of A1, A2a, and A3 suppressed testosterone production 20–50% testosterone production. Furthermore, Rp-cAMPS (cAMP antagonist) and Protein Kinase A (PKA) inhibitors (H89 and PKI) significantly decreased cordycepin-induced testosterone production, indicating that the PKA-cAMP signal pathway was activated by cordycepin through adenosine receptors. Moreover, cordycepin induced StAR protein expression, and H89 suppressed cordycepin-induced steroidogenic acute regulatory (StAR) protein expression. Conclusively, cordycepin associated with adenosine receptors to activate cAMP-PKA-StAR pathway and steroidogenesis in the mouse Leydig cells.

37-kDa Laminin Receptor Precursor Mediates GnRH-II–Induced MMP-2 Expression and Invasiveness in Ovarian Cancer Cells

GnRH-II enhances ovarian cancer cell invasion in an autocrine manner. We have now found that GnRH-II increases 37-kDa laminin receptor precursor (LRP) production in GnRH receptor (GnRHR)-positive OVCAR-3 and CaOV-3 ovarian cancer cells, while small interfering RNA (siRNA)-mediated depletion of GnRH-II or GnRHR mRNA abrogates this. The invasiveness of ovarian cancer cells is also reduced >85% by siRNA-mediated knockdown of LRP levels and >50% by pretreatment of Matrigel with a synthetic peptide that blocks interactions between laminin and the 67-kDa nonintegrin laminin receptor which comprises two LRP subunits. Conversely, overexpressing LRP in CaOV-3 cells increases their invasiveness 5-fold, while overexpressing LRP with a nonfunctional laminin-binding site does not. Depletion of LRP by siRNA treatment reduces CaOV-3 cell attachment to laminin-coated plates by ∼80% but only reduces their binding to Matrigel by ∼20%. Thus, while LRP influences CaOV-3 cell adhesion to laminin, LRP must act in other ways to enhance invasion. Matrix metalloproteinases (MMPs) are key mediators of invasion, and LRP siRNA treatment of OVCAR-3 and CaOV-3 cells inhibits MMP-2 but not MMP-9 mRNA levels. Overexpressing LRP in these cells increases MMP-2 production specifically, while a laminin-binding deficient LRP does not. Importantly, LRP siRNA treatment abolishes GnRH-II-induced MMP-2 production, and invasion in OVCAR-3 and CaOV-3 cells, which was also seen after MMP-2 siRNA treatment. These results suggest that GnRH-II-induced LRP expression increases the amount of the 67-kDa nonintegrin laminin receptor, which appears to interact with laminin in the extracellular matrix to promote MMP-2 expression and enhance ovarian cancer cell invasion.

Gonadotrophin-releasing hormone-I and -II stimulate steroidogenesis in prepubertal murine Leydig cells in vitro

AIM To study the effect and mechanism of gonadotrophin-releasing hormone (GnRH) on murine Leydig cell steroidogenesis. METHODS Purified murine Leydig cells were treated with GnRH-I and -II agonists, and testosterone production and steroidogenic enzyme expressions were determined. RESULTS GnRH-I and -II agonists significantly stimulated murine Leydig cell steroidogenesis 60%-80% in a dose- and time-dependent manner (P < 0.05). The mRNA expressions of steroidogenic acute regulatory (StAR) protein, P450scc, 3beta-hydroxysteroid dehydrogenase (HSD), but not 17alpha-hydroxylase or 17beta-HSD, were significantly stimulated by both GnRH agonists with a 1.5- to 3-fold increase (P < 0.05). However, only 3beta-HSD protein expression was induced by both GnRH agonists, with a 1.6- to 2-fold increase (P < 0.05). CONCLUSION GnRH directly stimulated murine Leydig cell steroidogenesis by activating 3b-HSD enzyme expression.

Gonadotropin-Releasing Hormone-II Increases Membrane Type I Metalloproteinase Production via β-Catenin Signaling in Ovarian Cancer Cells

GnRH-II is produced by ovarian cancer cells and enhances their invasiveness in vitro. In our studies of OVCAR-3 and CaOV-3 ovarian cancer cell lines, GnRH-II treatment induced phosphorylation of Akt and glycogen synthase kinase (GSK)3β, as well as β-catenin accumulation in the nucleus, and the latter was reduced by small interfering RNA (siRNA)-mediated depletion of the GnRH receptor. The phosphatidylinositol 3 kinase (PI3K)/Akt pathway is involved in β-catenin-dependent signaling, and pretreatment of these human ovarian cancer cells with a PI3K/Akt inhibitor, LY294002, attenuated GnRH-II-stimulated phosphorylation of GSK3β and inhibited GnRH-II-induced invasion. It also attenuated GnRH-II induced trans-activation of a β-catenin-dependent reporter gene, most likely because GSK3β phosphorylation promotes translocation of β-catenin to the nucleus. Membrane type I matrix metalloproteinase (MT1-MMP) contributes to tumor progression directly, or by processing the latent MMP-2 zymogen, and is a known target of β-catenin signaling. When OVCAR-3 and CaOV-3 cells were treated with GnRH-II, MT1-MMP levels increased approximately 3-fold, whereas siRNA-mediated depletion of GnRH receptor or pretreatment with LY294002 abrogated this. In addition, lithium chloride, which increases GSK3β phosphorylation and the nuclear translocation of β-catenin, increased MT1-MMP levels in these ovarian cancer cells. By contrast, depletion of β-catenin by siRNA treatment abolished GnRH-II-induced MT1-MMP synthesis and reduced their invasive potential. Furthermore, siRNA-mediated reduction of MT1-MMP levels reduced GnRH-II-induced invasion in ovarian cancer cells. We therefore conclude that GnRH-II stimulates the PI3K/Akt pathway, and the phosphorylation of GSK3β, thereby enhancing the β-catenin-dependent up-regulation of MT1-MMP production, which contributes to ovarian cancer metastasis.

Rapid Effect of GNRH1 on Follicle-Stimulating Hormone Beta Gene Expression in LbetaT2 Mouse Pituitary Cells Requires the Progesterone Receptor

Abstract Gonadotropin-releasing hormone (GNRH) activates the progesterone receptor (PGR) in pituitary cells and accentuates gonadotropin expression. We show that GNRH1 increases Fshb mRNA levels in LbetaT2 mouse pituitary cells within 8 h and is three times more effective than GNRH2. By contrast, GNRH1 and GNRH2 do not affect Lhb gene expression in these cells. Within the same time frame, small interfering RNA (siRNA) knockdown of the PGR in LbetaT2 cells reduced GNRH1 activation of a PGR response element (PRE)-driven luciferase reporter gene and Fshb mRNA levels by >50%. Chromatin immunoprecipitation (ChIP) assays also demonstrated that PGR loading on the PRE within the Fshb gene promoter in LbetaT2 cells occurred within 8 h after GNRH1 treatment and was lost by 24 h. While the GNRH1-induced upregulation of the PRE reporter gene and Fshb mRNA levels was attenuated by cotreatment with protein kinase A (H-89) and protein kinase C (GF109203X) inhibitors, only GF109203X inhibited PGR phosphorylation at Ser249 in LbetaT2 cells. Immunoprecipitation assays also showed a progressive increase in the interaction between the PGR and its coactivator NCOA3 that peaked at 8 h coincident with the increase in Fshb mRNA after GNRH1 treatment. The siRNA-mediated knockdown of NCOA3 in LbetaT2 cells also reduced Fshb mRNA levels after GNRH1 treatment and loading of NCOA3 on the Fshb promoter PRE in a ChIP assay. We conclude that the rapid effect of GNRH1 on Fshb expression in LbetaT2 cells is mediated by PGR phosphorylation and loading at the PRE within the Fshb promoter together with NCOA3.

THE EFFECTS OF TREMELLA AURANTIA ON TESTOSTERONE AND CORTICOSTERONE PRODUCTIONS IN NORMAL AND DIABETIC RATS

Tremella aurantia (TA) has been traditionally used as food and crude medicine in Chinese society. The polysaccharide isolated from the fruiting bodies of TA exhibits significant hypoglycemic activity in diabetic mouse models of insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM). Diabetes will cause sexual dysfunction in patients. In the present study, we examined if the treatment of TA on IDDM and NIDDM rats will restore steroidogenesis and then the reproductive function. The fruiting bodies (FB), mycelium (TM) and polysaccharide (GX) of TA were fed to the IDDM and NIDDM rats, and testosterone and corticosterone levels in plasma, the weight of steroidogenic organs, and the expression of steroidogenic acute regulatory (StAR) protein and P450scc enzyme were determined. Plasma testosterone productions were significantly suppressed with the feeding of FB or TM in normal rat (p < 0.05). Testosterone productions were also significantly suppressed in IDDM diabetes rats (p < 0.05), and FB or TM could not restore the inhibitory effects (p > 0.05). There was no significant difference of the testosterone production between normal and NIDDM rats (p > 0.05). In plasma corticosterone production, there were no differences among control, FB- or TM-fed normal rats (p > 0.05). Corticosterone levels were reduced in IDDM rats compared to control, and FB or TM could restore its level. Corticosterone levels were induced in NIDDM rats compared to control (p < 0.05), but FB, TM or GX significantly brought the corticosterone back (p < 0.05) to the control levels. Considering steroidogenic organs, IDDM rats with or without TA treatments had heavier testis and adrenal glands, but not epididymis, than normal rats with or without TA treatments. There were no effects of TA on the weight of steroidogenic organs among normal and NIDDM rats. However, GX feeding in NIDDM rat had lesser testis weight compared to NIDDM rats. The expression of StAR protein and P450scc enzyme were not different among groups in IDDM and NIDDM rats. Plasma testosterone productions were suppressed in normal rats with the feeding of TA (FB and TM). IDDM rats did have lower testosterone, but not in NIDDM, and FB or TM could not restore the inhibitory effects. The induction of IDDM or NIDDM rats did affect steroidogenesis and steroidogenic organ weights, and the feeding of TA had different effects on steroidogenesis in different types of diabetic rats.

Temporal Recruitment of Transcription Factors at the 3′,5′-Cyclic Adenosine 5′-Monophosphate-Response Element of the Human GnRH-II Promoter

GnRH-II is a potent GnRH subtype involved in modulating OVCAR-3 cell proliferation and the invasive properties of JEG-3 cells, and an atypical cAMP-response element (CRE) in the human GnRH-II promoter influences its activation. We demonstrated that the GnRH-II promoter is activated by 8-bromoadenosine-cAMP in several cell lines including alphaT3, TE671, JEG-3, and OVCAR-3 cells and that cAMP enhances GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Moreover, 8-bromoadenosine-cAMP increases cAMP response element-binding protein (CREB) phosphorylation in JEG-3 and OVCAR-3 cells and augments CBP and CCAAT/enhancer-binding protein (C/EBP)-beta coimmunoprecipitation with phosphorylated CREB (p-CREB) in a temporally defined manner from nuclear extracts. When CREB, CBP, and C/EBPbeta levels were knocked down by small interfering RNA, reductions in any of these transcription factors reduced cAMP-enhanced GnRH-II promoter activity and GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Importantly, chromatin immunoprecipitation assay showed that p-CREB bound the CRE within the endogenous GnRH-II promoter within 1 h and that p-CREB association with C/EBPbeta occurs within 2 h of cAMP stimulation, coincident with the first appearance of C/EBPbeta at the CRE. By contrast, maximum interactions between p-CREB and CBP do not occur until at least 4 h after cAMP stimulation, and this is reflected in the progressive loading of CBP at the CRE at 2-4 h, as demonstrated by chromatin immunoprecipitation. Taken together, these data suggest that p-CREB, C/EBPbeta, and CBP are recruited to the CRE of the GnRH-II promoter in a temporarily defined manner to enhance its transcription in JEG-3 and OVCAR-3 cells in response to cAMP.