T. John Wu

Targeted Deletion of the Vgf Gene Indicates that the Encoded Secretory Peptide Precursor Plays a Novel Role in the Regulation of Energy Balance

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

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.

LHRH-(1–5): a bioactive peptide regulating reproduction

Luteinizing hormone-releasing hormone-I (LHRH-I) was isolated from the mammalian hypothalamus and shown to be the primary regulator of reproduction through its initiation of pituitary gonadotropin release. Subsequently, it has also been shown to have non-pituitary actions. Although the regulation of LHRH-I synthesis and release has been extensively studied, there is additional evidence to suggest that processing of the peptide represents another layer of regulation. The focus of this review will be on evidence for the action of LHRH-(1-5), the pentapeptide metabolite of LHRH-I, in regulating LHRH-I synthesis, secretion and reproductive behavior. The involvement of LHRH-(1-5) in the control of aspects of reproduction might represent yet another level of regulatory complexity through neuropeptide processing.

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.

Screening for GPR101 defects in pediatric pituitary corticotropinomas

Cushing disease (CD) in children is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas. Germline or somatic mutations in genes such as MEN1, CDKIs, AIP, and USP8 have been identified in pediatric CD, but the genetic defects in a significant percentage of cases are still unknown. We investigated the orphan G protein-coupled receptor GPR101, a gene known to be involved in somatotropinomas, for its possible involvement in corticotropinomas. We performed GPR101 sequencing, expression analyses by RT-qPCR and immunostaining, and functional studies (cell proliferation, pituitary hormones secretion, and cAMP measurement) in a series of patients with sporadic CD secondary to ACTH-secreting adenomas in whom we had peripheral and tumor DNA (N=36). No increased GPR101 expression was observed in tumors compared to normal pituitary (NP) tissues, nor did we find a correlation between GPR101 and ACTH expression levels. Sequence analysis revealed a very rare germline heterozygous GPR101 variant (p.G31S) in one patient with CD. Overexpression of the p.G31S variant did not lead to increased growth and proliferation, although modest effects on cAMP signaling were seen. GPR101 is not overexpressed in ACTH-secreting tumors compared to NPs. A rare germline GPR101 variant was found in one patient with CD but in vitro studies did not support a consistent pathogenic effect. GPR101 is unlikely to be involved in the pathogenesis of CD.

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.