Darwin O. Larco

Postovariectomy weight gain in female rats is reversed by estrogen receptor α agonist, propylpyrazoletriol

OBJECTIVE The objective of this study was to distinguish the role of specific estrogen receptors (ERs), ERalpha and ERbeta, on body weight regulation using a rat model of weight gain subsequent to menopause. STUDY DESIGN Ovariectomized rats were utilized as the animal model to simulate the postmenopause weight gain. The rats were ovariectomized and subcutaneously injected daily with vehicle, estradiol-17beta (E2), propylpyrazoletriol (PPT; ERalpha agonist) and diarylpropionitrile (DPN; ERbeta agonist). To further control for the possible effect of estrogen secreted from adrenals, a second experiment was conducted during which the rats were adrenalectomized and ovariectomized. RESULTS Ovariectomy significantly increased (P < .05) body weight, whereas treatment of ovariectomized rats with E2 and PPT, but DPN decreased (P < .05) body weight. The results from the second study with ovariectomized/adrenalectomized rats were consistent with the first experiment. CONCLUSION These results suggest that the activation of ERalpha is important in regulating body weight.

GnRH-(1–5) Transactivates EGFR in Ishikawa Human Endometrial Cells via an Orphan G Protein-Coupled Receptor

The decapeptide GnRH is known for its central role in the regulation of the hypothalamo-pituitary-gonadal axis. In addition, it is also known to have local effects within peripheral tissues. The zinc metalloendopeptidase, EC 3.4.24.15 (EP24.15), can cleave GnRH at the Tyr(5)-Gly(6) bond to form the pentapeptide, GnRH-(1-5). The central and peripheral effect of GnRH-(1-5) is different from its parent peptide, GnRH. In the current study, we examined the effect of GnRH-(1-5) on epidermal growth factor receptor (EGFR) phosphorylation and cellular migration. Using the Ishikawa cell line as a model of endometrial cancer, we demonstrate that GnRH-(1-5) stimulates epidermal growth factor release, increases the phosphorylation of EGFR (P < .05) at three tyrosine sites (992, 1045, 1068), and promotes cellular migration. In addition, we also demonstrate that these actions of GnRH-(1-5) are mediated by the orphan G protein-coupled receptor 101 (GPR101). Down-regulation of GPR101 expression blocked the GnRH-(1-5)-mediated release of epidermal growth factor and the subsequent phosphorylation of EGFR and cellular migration. These results suggest that GPR101 is a critical requirement for GnRH-(1-5) transactivation of EGFR in Ishikawa cells.

The Metabolite GnRH-(1-5) Inhibits the Migration of Immortalized GnRH Neurons

The decapeptide GnRH is an important regulator of reproductive behavior and function. In the extracellular matrix, GnRH is metabolized by the endopeptidase EC3.4.24.15 (EP24.15) to generate the pentapeptide GnRH-(1-5). In addition to its expression in the adult hypothalamus, EP24.15 is expressed along the migratory path of GnRH-expressing neurons during development. Although we have previously demonstrated a role for EP24.15 in the generation of the biologically active pentapeptide GnRH-(1-5) in regulating GnRH expression and mediating sexual behavior during adulthood in rodents, the modulatory role of GnRH-(1-5) in the migration of GnRH neurons during development remains unknown. To address this information gap, we examined the effect of GnRH-(1-5) on the cellular migration of a premigratory GnRH-secreting neuronal cell line, the GN11 cell, using a wound-healing assay. Dose- and time-response studies demonstrated that GnRH-(1-5) significantly delayed wound closure. We then sought to identify the mechanism by which GnRH-(1-5) inhibits migration. Because the cognate GnRH receptor is a G protein-coupled receptor, we examined whether GnRH-(1-5) regulates migration by also activating a G protein-coupled receptor. Using a high-throughput β-arrestin recruitment assay, we identified an orphan G protein-coupled receptor (GPR173) that was specifically activated by GnRH-(1-5). Interestingly, small interfering RNA to GPR173 reversed the GnRH-(1-5)-mediated inhibition on migration of GN11 neurons. Furthermore, we also demonstrate that the GnRH-(1-5)-activated GPR173-dependent signal transduction pathway involves the activation of the signal transducer and activator of transcription 3 in GnRH migration. These findings indicate a potential regulatory role for GnRH-(1-5) in GnRH neuronal migration during development.

GnRH-(1-5) activates matrix metallopeptidase-9 to release epidermal growth factor and promote cellular invasion.

In the extracellular space, the gonadotropin-releasing hormone (GnRH) is metabolized by the zinc metalloendopeptidase EC3.4.24.15 (EP24.15) to form the pentapeptide, GnRH-(1-5). GnRH-(1-5) diverges in function and mechanism of action from GnRH in the brain and periphery. GnRH-(1-5) acts on the orphan G protein-coupled receptor 101 (GPR101) to sequentially stimulate epidermal growth factor (EGF) release, phosphorylate the EGF receptor (EGFR), and facilitate cellular migration. These GnRH-(1-5) actions are dependent on matrix metallopeptidase (MMP) activity. Here, we demonstrated that these GnRH-(1-5) effects are dependent on increased MMP-9 enzymatic activity in the Ishikawa and ECC-1 cell lines. Furthermore, the effects of GnRH-(1-5) mediated by GPR101 and the subsequent increase in MMP-9 enzymatic activity lead to an increase in cellular invasion. These results suggest that GnRH-(1-5) and GPR101 regulation of MMP-9 may have physiological relevance in the metastatic potential of endometrial cancer cells.

The Novel Actions of the Metabolite GnRH-(1-5) are Mediated by a G Protein-Coupled Receptor

The gonadotropin-releasing hormone (GnRH) was originally isolated from the mammalian hypothalamus for its role as the primary regulator of reproductive function. Since its discovery, GnRH has also been shown to be located in non-hypothalamic tissues and is known to have diverse functions. Although the regulation of GnRH synthesis and release has been extensively studied, there is additional evidence to suggest that the processing of GnRH to the metabolite GnRH-(1-5) represents another layer of regulation. The focus of this review will be on the current evidence for the action of the pentapeptide metabolite GnRH-(1-5) in regulating cellular migration. We discuss the potential role of GnRH-(1-5) in regulating GnRH neuronal migration during development. Furthermore, we demonstrate these actions are mediated by the activation of a G protein-coupled receptor. Our findings suggest that GnRH-(1-5) may play a developmental function in addition to regulating developing cells.

β-Arrestin 2 Is a Mediator of GnRH-(1–5) Signaling in Immortalized GnRH Neurons

We have previously demonstrated that the cleavage product of the full-length GnRH, GnRH-(1-5), is biologically active, binds G protein-coupled receptor 173 (GPR173), and inhibits the migration of cells in the immortalized GnRH-secreting GN11 cell. In this study, we attempted to characterize the GnRH-(1-5) intracellular signaling mechanism. To determine whether the signaling pathway mediating GnRH-(1-5) regulation of migration involves a G protein-dependent mechanism, cells were treated with a generic G protein antagonist in the presence and absence of GnRH-(1-5), and a wound-healing assay was conducted to measure migration. G Protein antagonist 2 treatment abolished the GnRH-(1-5) inhibition of migration, indicating that the mechanism of GnRH-(1-5) is G protein coupled. To identify the potential Gα-subunit recruited by GnRH-(1-5) binding GPR173, we measured the second messengers cAMP and inositol triphosphate levels. GnRH-(1-5) treatment did not alter cAMP levels relative to cells treated with vehicle or forskolin, suggesting that GnRH-(1-5) does not couple to the Gαs or Gαi subunits. Similarly, inositol triphosphate levels remained unchanged with GnRH-(1-5) treatment, indicating a mechanism not mediated by the Gαq/11 subunit. Therefore, we also examined whether GnRH-(1-5) activating GPR173 deviated from the canonical G protein-coupled receptor signaling pathway by coupling to β-arrestin 1/2 to regulate migration. Our coimmunoprecipitation studies indicate that GnRH-(1-5) induces the rapid interaction between GPR173 and β-arrestin 2 in GN11 cells. Furthermore, we demonstrate that this association recruits phosphatase and tensin homolog to mediate the downstream action of GnRH-(1-5). These findings suggest that the GnRH-(1-5) mechanism deviates from the canonical G protein-coupled receptor pathway to regulate cell migration in immortalized GnRH neurons.

The interaction of dietary isoflavones and estradiol replacement on behavior and brain-derived neurotrophic factor in the ovariectomized rat

Phytoestrogens are plant derived, non-steroidal compounds naturally found in rodent chows that potentially have endocrine-disrupting effects. Isoflavones, the most common phytoestrogens, have a similar structure and molecular weight to 17β-estradiol (E2) and have the ability to bind and activate both isoforms of the estrogen receptor (ER). Most isoflavones have a higher affinity for ERβ, which is involved in sexually dimorphic behavioral regulation. The goal of this study was to examine the interaction of isoflavones and E2 presence in the OVX rat on anxiety- and depressive- like behavior and the related BDNF pathophysiology. E2 administration resulted in anxiogenic behaviors when isoflavones were present in the diet (p<0.05), but anxiolytic behaviors when isoflavones were not present (p<0.05). E2 resulted in antidepressive-like behaviors in animals fed an isoflavone-rich diet (p<0.05), with no effect when isoflavones were removed. Increased hippocampal BDNF expression was observed in animals fed an isoflavone-rich diet after E2 administration (p<0.05). BDNF expression in the amygdala and hypothalamus was increased after E2 treatment in animals fed an isoflavone-rich diet. Overall, these results demonstrate that the presence of dietary isoflavones can differentially regulate the effect of E2 replacement on behavior and BDNF expression.

Dietary Isoflavone-Dependent and Estradiol Replacement Effects on Body Weight in the Ovariectomized (OVX) Rat

17β-Estradiol is known to regulate energy metabolism and body weight. Ovariectomy results in body weight gain while estradiol administration results in a reversal of weight gain. Isoflavones, found in rodent chow, can mimic estrogenic effects making it crucial to understand the role of these compounds on metabolic regulation. The goal of this study is to examine the effect of dietary isoflavones on body weight regulation in the ovariectomized rat. This study will examine how dietary isoflavones can interact with estradiol treatment to affect body weight. Consistent with previous findings, animals fed an isoflavone-rich diet had decreased body weight (p<0.05), abdominal fat (p<0.05), and serum leptin levels (p<0.05) compared to animals fed an isoflavone-free diet. Estradiol replacement resulted in decreased body weight (p<0.05), abdominal fat (p<0.05), and serum leptin (p<0.05). Current literature suggests the involvement of cytokines in the inflammatory response of body weight gain. We screened a host of cytokines and chemokines that may be altered by dietary isoflavones or estradiol replacement. Serum cytokine analysis revealed significant (p<0.05) diet-dependent increases in inflammatory cytokines (keratinocyte-derived chemokine). The isoflavone-free diet in OVX rats resulted in the regulation of the following cytokines and chemokines: interleukin-10, interleukin-18, serum regulated on activation, normal T cell expressed and secreted, and monocyte chemoattractant protein-1 (p<0.05). Overall, these results reveal that estradiol treatment can have differential effects on energy metabolism and body weight regulation depending on the presence of isoflavones in rodent chow.

GnRH-(1–5) Inhibits TGF-β Signaling to Regulate the Migration of Immortalized Gonadotropin-Releasing Hormone Neurons

Gonadotropin-releasing hormone (GnRH) neurons originate outside the central nervous system (CNS) in the nasal placode where their migration to the basal forebrain is dependent on the integration of multiple signaling cues during development. The proper migration and establishment of the GnRH neuronal population within the CNS are critical for normal pubertal onset and reproductive function. The endopeptidase EP24.15 is expressed along the migratory path of GnRH neurons and cleaves the full-length GnRH to generate the metabolite GnRH-(1–5). Using the GN11 cell model, which is considered a pre-migratory GnRH neuronal cell line, we demonstrated that GnRH-(1–5) inhibits cellular migration in a wound closure assay by binding the orphan G protein-coupled receptor 173 (GPR173). In our current experiments, we sought to utilize an in vitro migration assay that better reflects the external environment that migrating GnRH neurons are exposed to during development. Therefore, we used a transwell assay where the inserts were coated with or without a matrigel, a gelatinous mixture containing extracellular matrix (ECM) proteins, to mimic the extracellular environment. Interestingly, GnRH-(1–5) inhibited the ability of GN11 cells to migrate only through ECM mimetic and was dependent on GPR173. Furthermore, we found that GN11 cells secrete TGF-β1, 2, and 3 but only TGF-β1 release and signaling were inhibited by GnRH-(1–5). To identify potential mechanisms involved in the proteolytic activation of TGF-β, we measured a panel of genes implicated in ECM remodeling. We found that GnRH-(1–5) consistently increased tissue inhibitors of metalloproteinase 1 expression, which is an inhibitor of proteinase activity, leading to a decrease in bioactive TGF-β and subsequent signaling. These results suggest that GnRH-(1–5) activating GPR173 may modulate the response of migrating GnRH neurons to external cues present in the ECM environment via an autocrine-dependent mechanism involving TGF-β.

Abstract 944: The effects of GnRH-(1-5) on endometrial cancer cell lines

The Gonadotropin-Releasing Hormone (GnRH) is a central regulator of reproductive function and behavior. In the periphery, GnRH is secreted by gynecologic tissues to exert local effects. Furthermore, GnRH is processed in the extracellular environment by the metalloendopeptidase EP24.15 to generate the bioactive metabolite, GnRH-(1-5). We have previously demonstrated that GnRH-(1-5) transactivates the epidermal growth factor receptor (EGFR) pathway to promote cell migration by binding the orphan receptor GPR101 in the Ishikawa and ECC-1 endometrial cancer cell lines. In this study, we sought to determine whether the effects of GnRH-(1-5) were dependent on the stage of the endometrial cancer. Cell lines ACI-181, ACI-52, and ACI-80 derived from endometrial cancers representative of stages 1, 2, and 3 respectively were tested in their response to GnRH-(1-5) treatment. Furthermore, the expression of GPR101 was confirmed by western blot analysis. Treatment with 100nM GnRH-(1-5) did not have significant changes in phospho-EGFR (pEGFR) levels in all ACI cell lines. However, subsequent measurement of ERK phosphorylation demonstrated that ACI-181 cells treated with GnRH-(1-5) had significantly (p Funding Sources: This research was supported by grants from the John P. Murtha Cancer Center at Walter Reed National Military Medical Center through the Uniformed Services University and the National Science Foundation (IOS-1052288). Citation Format: Darwin O. Larco, Madelaine J. Cho-Clark, Maya Sorini, Cameron Lee, T. John Wu. The effects of GnRH-(1-5) on endometrial cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 944.