The Childrens Health Research Institute

London, Canada

The Childrens Health Research Institute

London, Canada
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Babwah A.V.,The Childrens Health Research Institute | Babwah A.V.,Lawson Health Research Institute | Babwah A.V.,University of Western Ontario | Navarro V.M.,The Childrens Health Research Institute | And 25 more authors.
Journal of Neuroscience | Year: 2015

The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility and kisspeptin (KP) is a potent trigger ofGnRHsecretion from GnRH neurons. KP signals via KISS1R, a Gαq/11-coupled receptor, and mice bearing a global deletion of Kiss1r (Kiss1r-/-) or a GnRH neuron-specific deletion of Kiss1r (Kiss1rd/d) display hypogonadotropic hypogonadism and infertility. KISS1R also signals via β-arrestin, and in mice lackingβ-arrestin-1 or-2, KP-triggered GnRH secretion is significantly diminished. Based on these findings, we hypothesized that ablation of Gαq/11 in GnRH neurons would diminish but not completely block KP-triggered GnRH secretion and that Gαq/11-independentGnRHsecretion would be sufficient to maintain fertility. To test this, Gnaq (encodesGaq) was selectively inactivated in the GnRH neurons of global Gna11 (encodes Gα11)-null mice by crossing Gnrh-Cre and Gnaqfl/fl;Gna11-/-mice. Experimental Gnaqfl/fl;Gna11-/-Gnrh-Cre (Gnaqd/d) and control Gnaqfl/fl;Gna11-/-(Gnaqfl/fl) littermate mice were generated and subjected to reproductive profiling. This process revealed that testicular development and spermatogenesis, preputial separation, and anogenital distance in males and day of vaginal opening and of first estrus in females were significantly less affected in Gnaqd/dmice than in previously characterized Kiss1r-/-or Kiss1rd/d mice. Additionally, Gnaqd/d males were subfertile, and although Gnaqd/d females did not ovulate spontaneously, they responded efficiently to a single dose of gonadotropins. Finally, KP stimulation triggered a significant increase in gonadotropins and testosterone levels in Gnaqd/d mice. We therefore conclude that the milder reproductive phenotypes and maintained responsiveness to KP and gonadotropins reflect Gαq/11-independent GnRH secretion and activation of the neuroendocrine-reproductive axis in Gnaqd/d mice. © 2015 the authors.

PubMed | The Childrens Health Research Institute and Lawson Health Research Institute
Type: | Journal: Reproductive biology and endocrinology : RB&E | Year: 2015

Expression of kisspeptin (protein) and Kiss1r (mRNA) was recently documented in the mouse uterus on D4 of pregnancy (the day of embryo implantation) suggesting that the uterine-based kisspeptin (KP)/kisspeptin receptor (KISS1R) signaling system regulates embryo implantation. Despite this important suggestion, it was never demonstrated that the uterus actually exhibits a functional KP/KISS1R signaling system on D4 of pregnancy. Thus, the goal of this study was to determine whether a functional KP/KISS1R signaling system exists in the mouse uterus on D4 of pregnancy.Since kisspeptin/KISS1R signaling triggers the phosphorylation of the mitogen-activated protein kinases p38 and ERK1/2, through immunohistochemical analyses, we determined whether exogenously administered kisspeptin could trigger p38 and ERK1/2 phosphorylation in the uterus on D4 of pregnancy. The results clearly demonstrated that kisspeptin could and that its effects were mediated via KISS1R. Additionally, the robust kisspeptin-triggered response was observed in the pregnant uterus only. Finally, it was demonstrated that on D4 of pregnancy the Kiss1 null uterus expresses functional KISS1R molecules capable of mediating the effects of kisspeptin.These results lead us to conclude that on D4 of pregnancy, the mouse uterus expresses a functional KP/KISS1R signaling system strengthening the possibility that this signaling system regulates embryo implantation. These findings strengthen the rationale for determining whether such a functional system exists in the uterus of the human female and if so, what role it might play in human pregnancy.

Re M.,The Childrens Health Research Institute | Re M.,Lawson Health Research Institute | Re M.,University of Western Ontario | Pampillo M.,The Childrens Health Research Institute | And 12 more authors.
PLoS ONE | Year: 2010

The mammalian type I gonadotropin releasing hormone receptor (GnRH-R) is a structurally unique G protein-coupled receptor (GPCR) that lacks cytoplasmic tail sequences and displays inefficient plasma membrane expression (PME). Compared to its murine counterparts, the primate type I receptor is inefficiently folded and retained in the endoplasmic reticulum (ER) leading to a further reduction in PME. The decrease in PME and concomitant increase in intracellular localization of the mammalian GnRH-RI led us to characterize the spatial distribution of the human and mouse GnRH receptors in two human cell lines, HEK 293 and HTR-8/SVneo. In both human cell lines we found the receptors were expressed in the cytoplasm and were associated with the ER and nuclear membrane. A molecular analysis of the receptor protein sequence led us to identify a putative monopartite nuclear localization sequence (NLS) in the first intracellular loop of GnRH-RI. Surprisingly, however, neither the deletion of the NLS nor the addition of the Xenopus GnRH-R cytoplasmic tail sequences to the human receptor altered its spatial distribution. Finally, we demonstrate that GnRH treatment of nuclei isolated from HEK 293 cells expressing exogenous GnRH-RI triggers a significant increase in the acetylation and phosphorylation of histone H3, thereby revealing that the nuclear-localized receptor is functional. Based on our findings, we conclude that the mammalian GnRH-RI is an intracellular GPCR that is expressed on the nuclear membrane. This major and novel discovery causes us to reassess the signaling potential of this physiologically and clinically important receptor. © 2010 Re et al.

Vo T.,University of Western Ontario | Vo T.,The Childrens Health Research Institute | Vo T.,Lawson Health Research Institute | Revesz A.,University of Western Ontario | And 11 more authors.
Journal of Endocrinology | Year: 2013

Epidemiological studies demonstrate that the link between impaired fetal development and glucose intolerance in later life is exacerbated by postnatal catch-up growth. Maternal protein restriction (MPR) during pregnancy and lactation in the rat has been previously demonstrated to lead to impaired glucose tolerance in adulthood, however the effects of protein restoration during weaning on glucose homeostasis are largely unknown. Recent in vitro studies have identified that the liver X receptor α(LXRα) maintains glucose homeostasis by inhibiting critical genes involved in gluconeogenesis including G6pase (G6pc), 11β-Hsd1 (Hsd11b1) and Pepck (Pck1). Therefore, we hypothesized that MPR with postnatal catch-up growth would impair LXRα in vivo, which in turn would lead to augmented gluconeogenic LXRα-target gene expression and glucose intolerance. To examine this hypothesis, pregnant Wistar rats were fed a control (20%) protein diet (C) or a low (8%) protein diet during pregnancy and switched to a control diet at birth (LP). At 4 months, the LP offspring had impaired glucose tolerance. In addition, LP offspring had decreased LXRα expression, while hepatic expression of 11β-HSD1 and G6Pase was significantly higher. This was concomitant with decreased binding of LXRα to the putative LXRE on 11β-Hsd1 and G6pase. Finally,we demonstrated that the acetylation of histone H3 (K9,14) surrounding the transcriptional start site of hepatic Lxrα (Nr1h3) was decreased in LP offspring, suggesting MPR-induced epigenetic silencing of the Lxrα promoter. In summary, our study demonstrates for the first time the important role of LXRα in mediating enhanced hepatic gluconeogenic gene expression and consequent glucose intolerance in adult MPR offspring. © 2013 Society for Endocrinology.

Pampillo M.,The Childrens Health Research Institute | Pampillo M.,Lawson Health Research Institute | Pampillo M.,University of Western Ontario | Babwah A.V.,The Childrens Health Research Institute | And 2 more authors.
Methods in Enzymology | Year: 2010

The kisspeptin/GPR54 signaling system positively regulates GnRH secretion, thereby acting as an important regulator of the hypothalamic-pituitary-gonadal axis. It also negatively regulates tumor metastases and placental trophoblast invasion. GPR54 is a Gq/11-coupled GPCR and activation by kisspeptin stimulates PIP2 hydrolysis and inositol phosphate (IP) formation, Ca2+ mobilization, arachidonic acid release, and ERK1/2 and p38 MAP kinase phosphorylation. Recently, we reported that GPR54 displays constitutive activity and internalization in the heterologous human embryonic kidney 293 cell system. Given the physiological and clinical importance of GPR54 as well as other GPCRs, we present assays for measuring constitutive receptor internalization and activity. Specifically, we describe the use of immunofluorescence coupled to confocal imaging, flow cytometry and indirect receptor radiolabeling to measure constitutive receptor internalization, and IP turnover in intact cells to measure constitutive activity. While we use the FLAG-tagged GPR54 molecule as an example to describe these assays, the assays can be applied to a wide range of GPCRs. © 2010 Elsevier Inc.

Szereszewski J.M.,The Childrens Health Research Institute | Szereszewski J.M.,Lawson Health Research Institute | Szereszewski J.M.,University of Western Ontario | Pampillo M.,The Childrens Health Research Institute | And 10 more authors.
PLoS ONE | Year: 2010

G protein-coupled receptor 54 (GPR54) is a Gq/11-coupled 7 transmembrane-spanning receptor (7TMR). Activation of GPR54 by kisspeptin (Kp) stimulates PIP2 hydrolysis, Ca2+ mobilization and ERK1/2 MAPK phosphorylation. Kp and GPR54 are established regulators of the hypothalamic-pituitary-gonadal (HPG) axis and loss-of-function mutations in GPR54 are associated with an absence of puberty and hypogonadotropic hypogonadism, thus defining an important role of the Kp/GPR54 signaling system in reproductive function. Given the tremendous physiological and clinical importance of the Kp/GPR54 signaling system, we explored the contributions of the GPR54-coupled Gq/11 and β-arrestin pathways on the activation of a major downstream signaling molecule, ERK, using Gq/11 and β-arrestin knockout mouse embryonic fibroblasts. Our study revealed that GPR54 employs the Gq/11 and β-arrestin-2 pathways in a co-dependent and temporally overlapping manner to positively regulate ERK activity and pERK nuclear localization. We also show that while β-arrestin-2 potentiates GPR54 signaling to ERK, β-arrestin-1 inhibits it. Our data also revealed that diminished β-arrestin-1 and -2 expression in the GT1-7 GnRH hypothalamic neuronal cell line triggered distinct patterns of gene expression following Kp-10 treatment. Thus, β-arrestin-1 and -2 also regulate distinct downstream responses in gene expression. Finally, we showed that GPR54, when uncoupled from the Gq/11 pathway, as is the case for several naturally occurring GPR54 mutants associated with hypogonadotropic hypogonadism, continues to regulate gene expression in a G protein-independent manner. These new and exciting findings add significantly to our mechanistic understanding of how this important receptor signals intracellularly in response to kisspeptin stimulation. © 2010 Szereszewski et al.

Taylor J.,The Childrens Health Research Institute | Taylor J.,Lawson Health Research Institute | Taylor J.,University of Western Ontario | Pampillo M.,The Childrens Health Research Institute | And 7 more authors.
Molecular Reproduction and Development | Year: 2014

During the first trimester of human pregnancy, cytotrophoblasts proliferate within the tips of the chorionic villi to form cell columns that anchor the placenta to the uterus. This migration coincides with a widespread change in the adhesion molecule repertoire of these trophoblasts. Kisspeptin and its receptor, KISS1R, are best known as potent triggers of gonadotropin-releasing hormone secretion. The kisspeptin/KISS1R signaling system is also highly expressed in the human placenta, where it was demonstrated to inhibit extra-villous trophoblast (EVT) migration and invasion in vitro. Here we show that kisspeptin, in a dose- and time-dependent manner, induces increased adhesion of human EVTs to type-I collagen, a major component of the human placenta. This increased adhesion was both rapid and transient, suggesting that it likely occurred through the activation of KISS1R secondary effectors such as PKC and ERK, which underwent rapid and transient kisspeptin-dependent activation in EVTs. We then showed that inhibition of both PKC and ERK1/2 attenuated the kisspeptin-dependent increase in EVT adhesion, suggesting that these molecules are key positive regulators of trophoblast adhesion. We therefore propose that kisspeptin/KISS1R signaling potentiates EVT adhesion to type-I collagen via "inside-out signaling." Furthermore, kisspeptin treatment increased mouse blastocyst adhesion to collagen I, suggesting that kisspeptin signaling is a key regulator of trophoblast function during implantation as well as early placentation. © 2013 Wiley Periodicals, Inc.

Sohi G.,University of Western Ontario | Sohi G.,The Childrens Health Research Institute | Sohi G.,Lawson Health Research Institute | Marchand K.,Lawson Health Research Institute | And 7 more authors.
Molecular Endocrinology | Year: 2011

Adverse events in utero, such as intrauterine growth restriction (IUGR), can permanently alter epigenetic mechanisms leading to the metabolic syndrome, which encompasses a variety of symptoms including augmented cholesterol. The major site for cholesterol homeostasis occurs via the actions of hepatic cholesterol 7α-hydroxylase (Cyp7a1), which catabolizes cholesterol to bile acids. To determine whether posttranslational histone modifications influence the long-term expression of Cyp7a1 in IUGR, we used a protein restriction model in rats. This diet during pregnancy and lactation led to IUGR offspring with decreased liver to body weight ratios, followed by increased circulating and hepatic cholesterol levels in both sexes at d 21 and exclusively in the male offspring at d 130. The augmented cholesterol was associated with decreases in the expression of Cyp7a1. Chromatin immunoprecipitation revealed that this was concomitant with diminished acetylation and enhanced methylation of histone H3 lysine 9 [K9,14], markers of chromatin silencing, surrounding the promoter region of Cyp7a1. These epigenetic modifications originate in part due to dietary-induced decreases in fetal hepatic Jmjd2a expression, a histone H3 [K9] demethylase. Collectively, these findings suggest that the augmented cholesterol observed in low-protein dietderived offspring is due to permanent repressive posttranslational histone modifications at the promoter of Cyp7a1. Moreover, this is the first study to demonstrate that maternal undernutrition leads to long-term cholesterol dysregulation in the offspring via epigenetic mechanisms. © 2011 by The Endocrine Society.

Goertzen C.G.,University of Western Ontario | Dragan M.,University of Western Ontario | Turley E.,University of Western Ontario | Babwah A.V.,University of Western Ontario | And 4 more authors.
Cellular Signalling | Year: 2016

Kisspeptins (KPs), peptide products of the KISS1 gene are endogenous ligands for the kisspeptin receptor (KISS1R), a G protein-coupled receptor. In numerous cancers, KISS1R signaling plays anti-metastatic roles. However, we have previously shown that in breast cancer cells lacking the estrogen receptor (ERα), kisspeptin-10 stimulates cell migration and invasion by cross-talking with the epidermal growth factor receptor (EGFR), via a β-arrestin-2-dependent mechanism. To further define the mechanisms by which KISS1R stimulates invasion, we determined the effect of down-regulating KISS1R expression in triple negative breast cancer cells. We found that depletion of KISS1R reduced their mesenchymal phenotype and invasiveness. We show for the first time that KISS1R signaling induces invadopodia formation and activation of key invadopodia proteins, cortactin, cofilin and membrane type I matrix metalloproteases (MT1-MMP). Moreover, KISS1R stimulated invadopodia formation occurs via a new pathway involving a β-arrestin2 and ERK1/2-dependent mechanism, independent of Src. Taken together, our findings suggest that targeting the KISS1R signaling axis might be a promising strategy to inhibit invasiveness and metastasis. © 2015.

Cvetkovic D.,University of Western Ontario | Babwah A.V.,University of Western Ontario | Babwah A.V.,The Childrens Health Research Institute | Babwah A.V.,Lawson Health Research Institute | And 2 more authors.
Journal of Cancer | Year: 2013

Kisspeptins (KP), peptide products of the kisspeptin-1 (KISSI) gene are the endogenous ligands for a G protein-coupled receptor (GPCR) - KP receptor (KISSIR). KISSIR couples to the Gaq/11 signaling pathway. KISSI is a metastasis suppressor gene and the KP/KISSIR signaling has anti-metastatic and tumor-suppressant effects in numerous human cancers. On the other hand, recent studies indicate that KP/KISSIR pathway plays detrimental roles in breast cancer. In this review, we summarize recent developments in the understanding of the mechanisms regulating KP/KISSIR signaling in breast cancer metastasis. © Ivyspring International Publisher.

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