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Fayazi M.,The Childrens Health Research Institute | Fayazi M.,Lawson Health Research Institute | Calder M.,The Childrens Health Research Institute | Calder M.,Lawson Health Research Institute | And 4 more authors.
Reproductive Biology and Endocrinology

Background: 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. Findings: 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. Conclusions: 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. © 2015 Fayazi et al. Source

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

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. Source

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

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. Source

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

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. Source

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.

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. Source

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