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Kisspeptinas , Neoplasias , Humanos , Kisspeptinas/metabolismo , Reproducción , Hormona Luteinizante/metabolismoRESUMEN
Nonalcoholic fatty liver disease (NAFLD), the most common liver disease, has become a silent worldwide pandemic. The incidence of NAFLD correlates with the rise in obesity, type 2 diabetes, and metabolic syndrome. A hallmark featureof NAFLD is excessive hepatic fat accumulation or steatosis, due to dysregulated hepatic fat metabolism, which can progress to nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. Currently, there are no approved pharmacotherapies to treat this disease. Here, we have found that activation of the kisspeptin 1 receptor (KISS1R) signaling pathway has therapeutic effects in NAFLD. Using high-fat diet-fed mice, we demonstrated that a deletion of hepatic Kiss1r exacerbated hepatic steatosis. In contrast, enhanced stimulation of KISS1R protected against steatosis in wild-type C57BL/6J mice and decreased fibrosis using a diet-induced mouse model of NASH. Mechanistically, we found that hepatic KISS1R signaling activates the master energy regulator, AMPK, to thereby decrease lipogenesis and progression to NASH. In patients with NAFLD and in high-fat diet-fed mice, hepatic KISS1/KISS1R expression and plasma kisspeptin levels were elevated, suggesting a compensatory mechanism to reduce triglyceride synthesis. These findings establish KISS1R as a therapeutic target to treat NASH.
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Diabetes Mellitus Tipo 2 , Enfermedad del Hígado Graso no Alcohólico , Animales , Diabetes Mellitus Tipo 2/metabolismo , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Humanos , Kisspeptinas/genética , Hígado/metabolismo , Cirrosis Hepática/patología , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Receptores de Kisspeptina-1/genética , Receptores de Kisspeptina-1/metabolismoRESUMEN
Embryo implantation failure is a major cause of infertility in women of reproductive age and a better understanding of uterine factors that regulate implantation is required for developing effective treatments for female infertility. This study investigated the role of the uterine kisspeptin receptor (KISS1R) in the molecular regulation of implantation in a mouse model. To conduct this study, a conditional uterine knockout (KO) of Kiss1r was created using the Pgr-Cre (progesterone receptor-CRE recombinase) driver. Reproductive profiling revealed that while KO females exhibited normal ovarian function and mated successfully to stud males, they exhibited significantly fewer implantation sites, reduced litter size and increased neonatal mortality demonstrating that uterine KISS1R is required for embryo implantation and a healthy pregnancy. Strikingly, in the uterus of Kiss1r KO mice on day 4 (D4) of pregnancy, the day of embryo implantation, KO females exhibited aberrantly elevated epithelial ERα (estrogen receptor α) transcriptional activity. This led to the temporal misexpression of several epithelial genes [Cftr (Cystic fibrosis transmembrane conductance regulator), Aqp5 (aquaporin 5), Aqp8 (aquaporin 8) and Cldn7 (claudin 7)] that mediate luminal fluid secretion and luminal opening. As a result, on D4 of pregnancy, the lumen remained open disrupting the final acquisition of endometrial receptivity and likely accounting for the reduction in implantation events. Our data clearly show that uterine KISS1R negatively regulates ERα signaling at the time of implantation, in part by inhibiting ERα overexpression and preventing detrimentally high ERα activity. To date, there are no reports on the regulation of ERα by KISS1R; therefore, this study has uncovered an important and powerful regulator of uterine ERα during early pregnancy.
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Implantación del Embrión , Células Epiteliales/metabolismo , Receptor alfa de Estrógeno/metabolismo , Regulación del Desarrollo de la Expresión Génica , Receptores de Kisspeptina-1/metabolismo , Transcripción Genética , Útero/metabolismo , Animales , Acuaporina 5/genética , Acuaporina 5/metabolismo , Acuaporinas/genética , Acuaporinas/metabolismo , Claudinas/genética , Claudinas/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Receptor alfa de Estrógeno/genética , Femenino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Embarazo , Receptores de Kisspeptina-1/genética , Transducción de Señal , Factores de TiempoRESUMEN
Triple-negative breast cancer (TNBC) is a highly metastatic and deadly disease. TNBC tumors lack estrogen receptor (ERα), progesterone receptor (PR), and HER2 (ErbB2) and exhibit increased glutamine metabolism, a requirement for tumor growth. The G protein-coupled kisspeptin receptor (KISS1R) is highly expressed in patient TNBC tumors and promotes malignant transformation of breast epithelial cells. This study found that TNBC patients displayed elevated plasma kisspeptin levels compared with healthy subjects. It also provides the first evidence that in addition to promoting tumor growth and metastasis in vivo, KISS1R-induced glutamine dependence of tumors. In addition, tracer-based metabolomics analyses revealed that KISS1R promoted glutaminolysis and nucleotide biosynthesis by increasing c-Myc and glutaminase levels, key regulators of glutamine metabolism. Overall, this study establishes KISS1R as a novel regulator of TNBC metabolism and metastasis, suggesting that targeting KISS1R could have therapeutic potential in the treatment of TNBC.
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Carcinogénesis/metabolismo , Reprogramación Celular , Metabolismo Energético , Receptores de Kisspeptina-1/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Adulto , Anciano , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Estudios de Casos y Controles , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Glutaminasa/genética , Glutaminasa/metabolismo , Glutamina/metabolismo , Humanos , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Invasividad Neoplásica , Nucleótidos/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores de Kisspeptina-1/genética , Transducción de Señal , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Carga Tumoral , Adulto JovenRESUMEN
The study investigated the effect of normal and supraphysiological (resulting from gonadotropin-dependent ovarian stimulation) levels of estradiol (E2) and progesterone (P4) on mouse uterine aquaporin gene/protein (Aqp/AQP) expression on Day 1 (D1) and D4 of pregnancy. The study also examined the effect of ovarian stimulation on uterine luminal closure and uterine receptivity on D4 of pregnancy and embryo implantation on D5 and D7 of pregnancy. These analyses revealed that the expression of Aqp3, Aqp4, Aqp5 and Aqp8 is induced by E2 while the expression of Aqp1 and Aqp11 is induced by P4. Additionally, P4 inhibits E2 induction of Aqp3 and Aqp4 expression while E2 inhibits Aqp1 and Aqp11 expression. Aqp9, however, is constitutively expressed. Ovarian stimulation disrupts Aqp3, Aqp5 and Aqp8 expression on D4 and AQP1, AQP3 and AQP5 spatial expression on both D1 and D4, strikingly so in the myometrium. Interestingly, while ovarian stimulation has no overt effect on luminal closure and uterine receptivity, it reduces implantation events, likely through a disruption in myometrial activity and embryo development. The wider implication of this study is that ovarian stimulation, which results in supraphysiological levels of E2 and P4 and changes (depending on the degree of stimulation) in the E2:P4 ratio, triggers abnormal expression of uterine AQP during pregnancy, and this is associated with implantation failure. These findings lead us to recognize that abnormal expression would also occur under any pathological state (such as endometriosis) that is associated with changes in the normal E2:P4 ratio. Thus, infertility among these patients might in part be linked to abnormal uterine AQP expression.
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Acuaporinas/fisiología , Implantación del Embrión/efectos de los fármacos , Estradiol/fisiología , Inducción de la Ovulación , Progesterona/fisiología , Animales , Acuaporinas/biosíntesis , Acuaporinas/genética , Implantación del Embrión/fisiología , Transferencia de Embrión , Estradiol/farmacología , Femenino , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Mifepristona/farmacología , Embarazo , Progesterona/farmacología , Seudoembarazo/metabolismo , Útero/fisiopatología , Agua/metabolismoRESUMEN
OBJECTIVE: To characterize the expression and signaling of uterine GPR83 in vivo in the nonpregnant and pregnant mouse and in vitro in human endometrial and nonendometrial cells. DESIGN: Controlled laboratory study. SETTING: Not applicable. PATIENTS: Not applicable. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Expression of uterine Gpr83 was determined by quantitative polymerase chain reaction throughout the estrous cycle and during early pregnancy in ovarian-stimulated and non-ovarian-stimulated mice and pregnant and pseudopregnant mice. Expression was also determined in ovariectomized mice after the administration of oil, E2, P4, or E2 + P4 and in stromal cells following 6 days of in vitro decidualization. GPR83 signaling was studied in human endometrial and embryonic kidney cell lines. Cells were treated by PEN, a GPR83 ligand, and PEN-induced extracellular signal-regulated kinase (ERK) phosphorylation was assayed under conditions that blocked Gαq/11 and/or ß-arrestin signaling. RESULTS: Uterine Gpr83 is expressed throughout the estrous cycle and during early pregnancy; expression increases dramatically at the time of uterine receptivity, embryo implantation, and stromal cell decidualization. In the ovariectomized mouse, hormone add-back reveals that Gpr83 expression is highly responsive to the combined treatment of E2 and P4, and studies in the ovarian-stimulated mouse show that expression is also very sensitive to changes in E2 and P4 and is therefore tightly regulated by E2 and P4. At the implantation site, expression is elevated up to D6 of pregnancy and then declines rapidly on D7 and D8, suggesting that if there is any involvement in decidualization, it is likely associated with primary but not secondary stromal cell decidualization. This premise was supported by the observation that stromal cell decidualization in vitro progresses with a decline in Gpr83 expression. In ERα/PR-expressing endometrial Ishikawa cells, GPR83 mediates PEN signals in a Gαq/11-dependent manner, and studies conducted in HEK 293 cells lacking ß-arrestin revealed that GPR83 also signals via a ß-arrestin-dependent manner. When signaling by either one or both pathways is downregulated, cells exhibit a major reduction in responsiveness to PEN treatment, demonstrating that signaling by both pathways is significant. CONCLUSION: We hypothesize that PEN/GPR83 signaling regulates uterine receptivity, embryo implantation, and primary stromal cell decidualization by coupling to Gαq/11- and ß-arrestin-dependent pathways.
RESUMEN
A nonreceptive uterus is a major cause of embryo implantation failure. This study examined the importance of the Gαq/11-coupled class of GPCRs as regulators of uterine receptivity. Mice were created lacking uterine Gαq and Gα11; as a result, signaling by all uterine Gαq/11-coupled receptors was disrupted. Reproductive profiling of the knockout females revealed that on d 4 of pregnancy, despite adequate serum progesterone (P4) levels and normal P4 receptor (PR) expression, there was no evidence of PR signaling. This resulted in the down-regulation of heart and neural crest derivatives expressed 2, Kruppel-like factor 15, and cyclin G1 and the subsequent persistent proliferation of the luminal epithelium. Aquaporin (Aqp) 11 was also potently down-regulated, whereas Aqp5/AQP5 expression persisted, resulting in the inhibition of luminal closure. Hypertrophy of the myometrial longitudinal muscle was also dramatically diminished, likely contributing to the observed implantation failure. Further analyses revealed that a major mechanism via which uterine Gαq/11 signaling induces PR signaling is through the transcriptional up-regulation of leucine-rich repeat-containing GPCR 4 (Lgr4). LGR4 was previously identified as a trigger of PR activation and signaling. Overall, this study establishes that Gαq/11 signaling, in a P4-dependent manner, critically regulates the acquisition of uterine receptivity in the female mouse, and disruption of such signaling results in P4 resistance.-de Oliveira, V., Schaefer, J., Calder, M., Lydon, J. P., DeMayo, F. J., Bhattacharya, M., Radovick, S., Babwah, A. V. Uterine Gαq/11 signaling, in a progesterone-dependent manner, critically regulates the acquisition of uterine receptivity in the female mouse.
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Implantación del Embrión/fisiología , Progesterona/sangre , Receptores de Progesterona/metabolismo , Útero/metabolismo , Animales , Acuaporinas/genética , Acuaporinas/metabolismo , Implantación del Embrión/genética , Femenino , Expresión Génica , Ratones , Miometrio/metabolismo , Embarazo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Progesterona/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Kisspeptins (KPs), peptide products of the kisspeptin-1 (KISS1) gene, are the endogenous ligands for the KISS1 receptor, KISS1R, which is a G protein-coupled receptor. In many human tumors, KISS1 functions as a metastasis-suppressor gene and KISS1/KISS1R signaling has antimetastatic and tumor-suppressor roles. On the contrary, emerging evidence indicates that the KP/KISS1R pathway plays detrimental roles in triple negative breast cancer (TNBC), the most difficult type of breast cancer to treat. TNBC patients initially respond to chemotherapy, but tumors acquire drug resistance and many patients relapse and die of metastases within a few years. In this review, we summarize recent developments in the understanding of the mechanisms by which KP/KISS1R signaling plays an adverse role in TNBC. This includes focusing on how KISS1R signaling regulates the cell cytoskeleton to induce tumor invadopodia formation and how KISS1R communicates with growth factor receptors such as the epidermal growth factor receptor, the receptor tyrosine kinase AXL, and transforming growth factor-ß to promote cell invasion, metastasis, and drug resistance.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Kisspeptinas/fisiología , Recurrencia Local de Neoplasia/genética , Receptores de Kisspeptina-1/fisiología , Animales , Femenino , Humanos , Kisspeptinas/genética , Metástasis de la Neoplasia , Receptores de Kisspeptina-1/genética , Transducción de Señal/genéticaRESUMEN
The KISS1 gene encodes KISS1, a protein that is rapidly processed in serum into smaller but biologically active peptides called kisspeptins (KPs). KISS1 and the KPs signal via the G-protein coupled receptor KISS1R. While KISS1 and KPs are recognized as potent positive regulators of the reproductive neuroendocrine axis in mammals, the first reported role for KISS1 was that of metastasis suppression in melanoma. Since then, it has become apparent that KISS1, KPs, and KISS1R regulate the development and progression of several cancers but interestingly, while these molecules act as suppressors of tumorigenesis and metastasis in many cancers, in breast and liver cancer they function as promoters. Thus, they join a small but growing number of molecules that exhibit dual roles in cancer highlighting the importance of studying cancer in context. Given their roles, KISS1, KPs and KISS1R represent important molecules in the development of novel therapies and/or as prognostic markers in treating cancer. However, getting to that point requires a detailed understanding of the relationship between these molecules and different cancers. The purpose of this review is therefore to highlight and discuss the clinical studies that have begun describing this relationship in varying cancer types including breast, liver, pancreatic, colorectal, bladder, and ovarian. An emerging theme from the reviewed studies is that the relationship between these molecules and a given cancer is complex and affected by many factors such as the micro-environment and steroid receptor status of the cancer cell. Our review and discussion of these important clinical studies should serve as a valuable resource in the successful development of future clinical studies.
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Breast cancer is a leading cause of cancer mortality. In particular, triple negative breast cancer (TNBC) comprise a heterogeneous group of basal-like tumors lacking estrogen receptor (ERα), progesterone receptor (PR) and HER2 (ErbB2). TNBC represents 15-20% of all breast cancers and occurs frequently in women under 50 years of age. Unfortunately, these patients lack targeted therapy, are typically high grade and metastatic at time of diagnosis. The mechanisms regulating metastasis remain poorly understood. We have previously shown that the kisspeptin receptor, KISS1R stimulates invasiveness of TNBC cells. In this report, we demonstrate that KISS1R signals via the secreted extracellular matrix protein, fibulin-3, to regulate TNBC invasion. We found that the fibulin-3 gene is amplified in TNBC primary tumors and that plasma fibulin-3 levels are elevated in TNBC patients compared to healthy subjects. In this study, we show that KISS1R activation increases fibulin-3 expression and secretion. We show that fibulin-3 regulates TNBC metastasis in a mouse experimental metastasis xenograft model and signals downstream of KISS1R to stimulate TNBC invasion, by activating matrix metalloproteinase 9 (MMP-9) and the MAPK pathway. These results identify fibulin-3 as a new downstream mediator of KISS1R signaling and as a potential biomarker for TNBC progression and metastasis, thus revealing KISS1R and fibulin-3 as novel drug targets in TNBC.
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Triple-negative breast cancer (TNBC) lacks the expression of estrogen receptor α, progesterone receptor and human epidermal growth factor receptor 2 (HER2). TNBC patients lack targeted therapies, as they fail to respond to endocrine and anti-HER2 therapy. Prognosis for this aggressive cancer subtype is poor and survival is limited due to the development of resistance to available chemotherapies and resultant metastases. The mechanisms regulating tumor resistance are poorly understood. Here we demonstrate that the G protein-coupled kisspeptin receptor (KISS1R) promotes drug resistance in TNBC cells. KISS1R binds kisspeptins, peptide products of the KISS1 gene and in numerous cancers, this signaling pathway plays anti-metastatic roles. However, in TNBC, KISS1R promotes tumor invasion. We show that KISS1 and KISS1R mRNA and KISS1R protein are upregulated in TNBC tumors, compared to normal breast tissue. KISS1R signaling promotes drug resistance by increasing the expression of efflux drug transporter, breast cancer resistance protein (BCRP) and by inducing the activity and transcription of the receptor tyrosine kinase, AXL. BCRP and AXL transcripts are elevated in TNBC tumors, compared to normal breast, and TNBC tumors expressing KISS1R also express AXL and BCRP. Thus, KISS1R represents a potentially novel therapeutic target to restore drug sensitivity in TNBC patients.
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Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Receptores de Kisspeptina-1/biosíntesis , Neoplasias de la Mama Triple Negativas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/biosíntesis , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Línea Celular Tumoral , Femenino , Humanos , Kisspeptinas/biosíntesis , Kisspeptinas/genética , Invasividad Neoplásica , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Kisspeptina-1/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Tirosina Quinasa del Receptor AxlRESUMEN
A major intracellular calcium (Ca2+) uptake pathway in both excitable and non-excitable eukaryotic cells is store-operated Ca2+ entry (SOCE). SOCE is the process by which endoplasmic reticulum (ER)-stored Ca2+ depletion leads to activation of plasma membrane Ca2+ channels to provide a sustained increase in cytosolic Ca2+ levels that mediate a plethora of physiological processes ranging from the immune response to platelet aggregation. Stromal interaction molecule-1 (STIM1) is the principal regulator of SOCE and responds to changes in ER stored Ca2+ through luminal sensing machinery composed of EF-hand and SAM domains (EFSAM). The EFSAM domain can undergo N-glycosylation at Asn131 and Asn171 sites; however, the precise role of EFSAM N-glycosylation in the Ca2+ sensing mechanism of STIM1 is unclear. By establishing a site-specific chemical approach to covalently linking glucose to EFSAM and examining α-helicity, thermal stability, three dimensional atomic-resolution structure, Ca2+ binding affinity and oligomerization, we show that N-glycosylation of the EFSAM domain enhances the properties that promote STIM1 activation. This augmentation occurs through changes in structure localized near the Asn131 and Asn171 sites that together permeate through the protein core and lead to decreased Ca2+ binding affinity, reduced stability and enhanced oligomerization. Congruently, Ca2+ influx via SOCE in HEK293 cells co-expressing Orai1 and STIM1 was diminished when N-glycosylation was blocked by introducing Asn131Gln and Asn171Gln mutations. Collectively, our data suggests that N-glycosylation enhances the EFSAM destabilization-coupled oligomerization in response to ER Ca2+ depletion thereby augmenting the role of STIM1 as a robust ON/OFF regulator of SOCE. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
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Asparagina/metabolismo , Proteínas de Neoplasias/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Animales , Asparagina/química , Dicroismo Circular , Glicosilación , Células HEK293 , Humanos , Espectroscopía de Resonancia Magnética , Espectrofotometría UltravioletaRESUMEN
Uterine growth and endometrial gland formation (adenogenesis) and function, are essential for fertility and are controlled by estrogens and other regulators, whose nature and physiological relevance are yet to be elucidated. Kisspeptin, which signals via Kiss1r, is essential for fertility, primarily through its central control of the hypothalamic-pituitary-ovarian axis, but also likely through peripheral actions. Using genetically modified mice, we addressed the contributions of central and peripheral kisspeptin signaling in regulating uterine growth and adenogenesis. Global ablation of Kiss1 or Kiss1r dramatically suppressed uterine growth and almost fully prevented adenogenesis. However, while uterine growth was fully rescued by E2 treatment of Kiss1(-/-) mice and by genetic restoration of kisspeptin signaling in GnRH neurons in Kiss1r(-/-) mice, functional adenogenesis was only marginally restored. Thus, while uterine growth is largely dependent on ovarian E2-output via central kisspeptin signaling, peripheral kisspeptin signaling is indispensable for endometrial adenogenesis and function, essential aspects of reproductive competence.
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Endometrio/crecimiento & desarrollo , Kisspeptinas/genética , Receptores de Kisspeptina-1/genética , Útero/crecimiento & desarrollo , Animales , Endometrio/metabolismo , Estrógenos/genética , Femenino , Hormona Liberadora de Gonadotropina/genética , Hormona Liberadora de Gonadotropina/metabolismo , Ratones , Neuronas/metabolismo , Organogénesis/genética , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Reproducción/genética , Reproducción/fisiología , Transducción de Señal/genética , Útero/metabolismoRESUMEN
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 ß-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 ß-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.
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Arrestinas/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Podosomas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Butadienos/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Cofilina 1/metabolismo , Cortactina/metabolismo , Receptores ErbB/metabolismo , Femenino , Humanos , Metaloproteinasa 14 de la Matriz/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Nitrilos/farmacología , Fosforilación/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Receptores de Kisspeptina-1 , Transducción de Señal/efectos de los fármacos , Arrestina beta 2 , beta-ArrestinasRESUMEN
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.
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Implantación del Embrión/fisiología , Kisspeptinas/fisiología , Receptores Acoplados a Proteínas G/fisiología , Transducción de Señal/fisiología , Útero/fisiología , Animales , Femenino , Ratones , Ratones Noqueados , Embarazo , Receptores de Kisspeptina-1RESUMEN
The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility and kisspeptin (KP) is a potent trigger of GnRH secretion 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 (Kiss1r(d/d)) display hypogonadotropic hypogonadism and infertility. KISS1R also signals via ß-arrestin, and in mice lacking ß-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-independent GnRH secretion would be sufficient to maintain fertility. To test this, Gnaq (encodes Gαq) was selectively inactivated in the GnRH neurons of global Gna11 (encodes Gα11)-null mice by crossing Gnrh-Cre and Gnaq(fl/fl);Gna11(-/-) mice. Experimental Gnaq(fl/fl);Gna11(-/-);Gnrh-Cre (Gnaq(d/d)) and control Gnaq(fl/fl);Gna11(-/-) (Gnaq(fl/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 Gnaq(d/d) mice than in previously characterized Kiss1r(-/-) or Kiss1r(d/d) mice. Additionally, Gnaq(d/d) males were subfertile, and although Gnaq(d/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 Gnaq(d/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 Gnaq(d/d) mice. SIGNIFICANCE STATEMENT: The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility. Over the last decade, several studies have established that the KISS1 receptor, KISS1R, is a potent trigger of GnRH secretion and inactivation of KISS1R on the GnRH neuron results in infertility. While KISS1R is best understood as a Gαq/11-coupled receptor, we previously demonstrated that it could couple to and signal via non-Gαq/11-coupled pathways. The present study confirms these findings and, more importantly, while it establishes Gαq/11-coupled signaling as a major conduit of GnRH secretion, it also uncovers a significant role for non-Gαq/11-coupled signaling in potentiating reproductive development and function. This study further suggests that by augmenting signaling via these pathways, GnRH secretion can be enhanced to treat some forms of infertility.
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Subunidades alfa de la Proteína de Unión al GTP/deficiencia , Hormona Liberadora de Gonadotropina/fisiología , Hipogonadismo/fisiopatología , Infertilidad Femenina/fisiopatología , Infertilidad Masculina/fisiopatología , Animales , Blastocisto/patología , Desarrollo Embrionario , Femenino , Subunidades alfa de la Proteína de Unión al GTP/fisiología , Perfilación de la Expresión Génica , Genitales Femeninos/patología , Genitales Femeninos/fisiopatología , Genitales Masculinos/patología , Genitales Masculinos/fisiopatología , Hormonas Esteroides Gonadales/metabolismo , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Gonadotropinas Hipofisarias/metabolismo , Gonadotropinas Hipofisarias/farmacología , Hipogonadismo/genética , Hipogonadismo/patología , Sistema Hipotálamo-Hipofisario/fisiopatología , Hipotálamo/patología , Infertilidad Femenina/embriología , Infertilidad Femenina/genética , Infertilidad Masculina/embriología , Infertilidad Masculina/genética , Kisspeptinas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Oligopéptidos/farmacología , Ovariectomía , Ovulación/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Péptidos/farmacología , Fenotipo , Receptores Acoplados a Proteínas G , Receptores de Kisspeptina-1 , EspermatogénesisRESUMEN
The hypothalamic-based kisspeptin-signaling system is a major positive regulator of the neuroendocrine-reproductive axis in mammals. During the last decade, major advances have been made in understanding how this signaling system is regulated and how it can be manipulated clinically to achieve beneficial outcomes in treating sex steroid-dependent disorders. Interestingly, kisspeptin was not first identified as a regulator of fertility. Instead, approximately 7 years earlier KISS1 was reported to be expressed in nonmetastatic melanoma cells and was subsequently demonstrated to act as a powerful suppressor of the metastatic potential of malignant melanoma cells. Since this discovery, numerous studies have demonstrated the expression of the kisspeptin-signaling system at several peripheral sites implicating it in biological processes such as the regulation of ovarian function, embryo implantation, placentation, angiogenesis, insulin secretion, and kidney development. Although much work remains to be done to assess how important kisspeptin signaling is in regulating some of these processes, for other processes recent studies have made tremendous strides toward such an assessment. Using mice lacking either Kiss1 or Kiss1r alleles, researchers have provided compelling evidence for kisspeptin playing a major role in regulating breast cancer metastasis, oocyte survival, follicular maturation, ovulation, and embryo implantation. This review critically discusses the findings from these as well as other studies which suggest roles for kisspeptin in regulating important physiological processes beyond the brain. It also discusses the challenges that lie ahead in determining whether findings made with animal models are relevant in humans.
Asunto(s)
Encéfalo/metabolismo , Kisspeptinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Reproducción/fisiología , Animales , Femenino , Fertilidad/fisiología , Kisspeptinas/genética , Masculino , Ratones , Ratones Noqueados , Ovulación/fisiología , Receptores Acoplados a Proteínas G/genética , Receptores de Kisspeptina-1 , Transducción de Señal/fisiologíaRESUMEN
Hypothalamic GnRH is the master regulator of the neuroendocrine reproductive axis, and its secretion is regulated by many factors. Among these is kisspeptin (Kp), a potent trigger of GnRH secretion. Kp signals via the Kp receptor (KISS1R), a Gαq/11-coupled 7-transmembrane-spanning receptor. Until this study, it was understood that KISS1R mediates GnRH secretion via the Gαq/11-coupled pathway in an ERK1/2-dependent manner. We recently demonstrated that KISS1R also signals independently of Gαq/11 via ß-arrestin and that this pathway also mediates ERK1/2 activation. Because GnRH secretion is ERK1/2-dependent, we hypothesized that KISS1R regulates GnRH secretion via both the Gαq/11- and ß-arrestin-coupled pathways. To test this hypothesis, we measured LH secretion, a surrogate marker of GnRH secretion, in mice lacking either ß-arrestin-1 or ß-arrestin-2. Results revealed that Kp-dependent LH secretion was significantly diminished relative to wild-type mice (P < .001), thus supporting that ß-arrestin mediates Kp-induced GnRH secretion. Based on this, we hypothesized that Gαq/11-uncoupled KISS1R mutants, like L148S, will display Gαq/11-independent signaling. To test this hypothesis, L148S was expressed in HEK 293 cells. and results confirmed that, although strongly uncoupled from Gαq/11, L148S retained the ability to trigger significant Kp-dependent ERK1/2 phosphorylation (P < .05). Furthermore, using mouse embryonic fibroblasts lacking ß-arrestin-1 and -2, we demonstrated that L148S-mediated ERK1/2 phosphorylation is ß-arrestin-dependent. Overall, we conclude that KISS1R signals via Gαq/11 and ß-arrestin to regulate GnRH secretion. This novel and important finding could explain why patients bearing some types of Gαq/11-uncoupled KISS1R mutants display partial gonadotropic deficiency and even a reversal of the condition, idiopathic hypogonadotropic hypogonadism.