No description available.

This study was designed to examine the effect of genistein, a botanical derived primarily from legumes, on pancreatic β-cell proliferation and the related molecular mechanisms. Diabetes mellitus is a major and growing public health problem worldwide. Both in type 1 (T1D) and type 2 diabetes (T2D), the deterioration of glycemic control over time is primarily caused by an inadequate mass and progressive dysfunction of β-cells. Therefore, the search for novel, safe and cost-effective agents that can enhance islet β-cell proliferation, thereby preserving β-cell mass, could be one of the essential strategies to prevent diabetes, given that β-cells have the potential to regenerate by proliferation of pre-existing b-cells in both physiological condition and after onset of diabetes. Genistein has various biological actions. However, studies on whether genistein has an effect on pancreatic β-cell function are very limited. Our laboratory recently found that genistein activates cAMP/protein kinase A (PKA) signaling in both clonal β-cells and mouse islets. Here I present evidence that genistein induced cellular proliferation of clonal rat pancreatic β-cells (INS1) and human islets following 24 h of incubation. This effect was dose-dependent with 5 µM genistein inducing a maximal 41% increase. The effect of genistein on cell proliferation was not dependent on estrogen receptors because this effect was not blocked by the estrogen receptor inhibitor ICI182,780. In addition, the genistein effect on β-cell proliferation was not shared by 17-β-estradiol or a host of structurally related flavonoid compounds, suggesting that this genistein action is structure-specific. Pharmacological or molecular intervention of PKA or MEK1/2, the upstream kinase of p42/44 mitogen activated protein kinases (ERK1/2), completely abolished the genistein-stimulated proliferation of INS1 cells and human islets, suggesting that both molecules are essential for genistein action. Consistent with its effect on cell proliferation, genistein increased intracellular cAMP and subsequently activated PKA in human islets. Genistein also caused a rapid and sustained phosphorylation of ERK1/2 with a maximal increase of 185% at 5 µM genistein. The genistein-induced ERK1/2 activation was completely ablated by inhibition of PKA in INS1 cells and human islets. Furthermore, I found that genistein induced protein expression of cyclin D1, a nuclear target of PKA and ERK1/2 activation and a major cell-cycle regulator essential for ï ¢-cell growth. These findings demonstrated that genistein may be a plant-derived growth factor for pancreatic β-cells involving induction of cyclin D1 via activation of the cAMP/PKA-dependent ERK1/2 signaling pathway, thereby providing a novel role for genistein in the regulation of pancreatic β-cell function. / Master of Science

Yes / Group I metabotropic glutamate receptor (mGluR) dependent long-term depression (LTD) is a major form of synaptic plasticity underlying learning and memory. The molecular mechanisms involved in mGluR-LTD have been investigated intensively for the last two decades. In this 60th anniversary special issue article, we review the recent advances in determining the mechanisms that regulate the induction, transduction and expression of mGluR-LTD in the hippocampus, with a focus on the mitogen-activated protein kinase (MAPK) pathways. In particular we discuss the requirement of p38 MAPK and extracellular signal-regulated kinase 1/2 (ERK 1/2) activation. The recent advances in understanding the signaling cascades regulating mGluR-LTD are then related to the cognitive impairments observed in neurological disorders, such as fragile X syndrome and Alzheimer's disease.

Le développement et l’homéostasie cellulaire de la prostate impliquent le contrôle strict des voies de signalisation induites par les androgènes et les facteurs de croissance. Ces diverses voies sont profondément altérées dans le cancer de la prostate, notamment lors des stades les plus avancés. Dans ce travail, une lignée immortalisée à partir de l’épithélium de prostate humaine, la lignée RWPE-1, a été utilisée pour étudier certains signaux régulant la prolifération cellulaire, ainsi que les connexions entre les voies de signalisation correspondantes. La prolifération des cellules RWPE-1 est sous la dépendance de l’EGF (Epidermal Growth Factor) qui intervient physiologiquement dans le développement épithélial. Les récepteurs apparentés à l’EGF-R sont également impliqués dans la prolifération au cours de la progression tumorale. La prolifération des cellules RWPE-1 en réponse à l’EGF est strictement dépendante de la voie ERK1/2, qui est donc considérée comme un point d’intégration des signaux. L’utilisation d’inhibiteurs du récepteur aux androgènes a permis de montrer le rôle essentiel qu’il joue dans l’activation d’ERK1/2 en réponse à l’EGF. Le récepteur aux androgènes s’associe avec plusieurs molécules de signalisation dans les cellules RWPE-1. Je démontre ici pour la première fois une association entre le récepteur aux androgènes et la kinase Raf-1, activatrice de la voie ERK1/2. Ainsi, le récepteur aux androgènes contrôlerait directement un processus essentiel à la prolifération épithéliale selon un mode d’action non-génomique. Par ailleurs, j’ai montré que la réponse proliférative des cellules RWPE-1 à l’IL-6 requiert l’activation de la voie ERK1/2, et l’activité kinase de l’EGF-R, suggérant la transactivation de ce récepteur par l’IL-6. L’utilisation de divers inhibiteurs chimiques a permis de démontrer que les métalloprotéases de la famille ADAM (a disintegrin and metalloprotease), notamment ADAM17, sont impliquées dans ce processus. Ainsi, l’activation de protéines ADAM par l’IL-6 conduirait au clivage d’un ligand membranaire de l’EGF-R, aboutissant à l’activation de la voie ERK1/2. Ce nouveau mécanisme pourrait être impliqué dans les situations inflammatoires conduisant à une prolifération excessive de l’épithélium prostatique, prélude à la transformation tumorale. En conclusion, les voies de signalisation étudiées sont fortement connectées dans les cellules épithéliales normales. Les deux nouveaux mécanismes décrits ici aboutissent à l’activation des kinases ERK1/2, point d’intégration et de convergence des voies de signalisation dans les cellules épithéliales de prostate normale. / Prostate development and cell homeostasis involve strict control of androgen and growth factors induced signaling pathways. These signaling pathways are deeply altered in prostate cancer, especially during late stages. In this work, the RWPE-1 immortalized cell line derived from human prostate epithelium has been used to study the signaling pathways regulating cell proliferation and their crosstalk. Optimal RWPE-1 proliferation is dependent on EGF (Epidermal Growth Factor), that also controls normal epithelial development. EGF-R family is also involved in cancer cell proliferation. EGF-dependent RWPE- 1 cell proliferation relies strictly on the ERK1/2 pathway which is then seen as a signal integrating node. Specific inhibitors showed essential role of androgen receptor in EGF mediated ERK1/2 activation. Androgen receptor is associated with several signaling molecules in RWPE-1 cells. I show here for the first time the physical interaction between the androgen receptor and the ERK1/2 activating kinase Raf1. Then, the androgen receptor could directly regulate an essential pathway for epithelial cells proliferation through a non-genomic mechanism. In addition, I showed that IL-6 dependent RWPE-1 cells proliferation requires both ERK1/2 and EGF-R kinase activities, suggesting an IL-6 mediated transactivation of EGF-R. By using several inhibitors, I showed that ADAM (a disintegrin and metalloprotease) family metaloproteases, especialy ADAM17, are involved in this process. IL-6-mediated ADAM proteins activation could lead to the cleavage of a membrane bound EGF-R ligand, leading to ERK1/2 pathway activation. This new mechanism could be involved in the inflammatory situations inducing hyperproliferation of the prostate epithelium, the first step of the transformation process. To conclude, the signaling pathways I studied are strongly connected in normal epithelial cells. The two new mechanisms described in this study lead to ERK1/2 kinases activation, an integrating node of signaling pathways in normal prostate epithelial cells.

Un remodelage vasculaire anormal est à la base de la pathogenèse des maladies cardio-vasculaires (MCV) telles que l’athérosclérose et l’hypertension. Des dysfonctionnements au niveau de la migration, l’hypertrophie et la prolifération des cellules musculaires lisses vasculaires (CMLV) sont des évènements cellulaires qui jouent un rôle primordial dans le remodelage vasculaire. L’insulin-like growth factor 1 (IGF-1), puissant facteur mitogène, contribue au développement des MCV, notamment via l’activation des protéines MAPK et PI3-K/PKB, composantes clés impliquées dans les voies de croissance cellulaire. Ces molécules sont également impliquées dans la modulation de l’expression de nombreux facteurs de transcription, incluant le facteur Egr-1. Egr-1 est régulé à la hausse dans différents types de maladies vasculaires impliquant les voies de signalisation de croissance et de stress oxydant qui par ailleurs peuvent être déclenchées par l’IGF-1. Cependant, la question d’une possible modulation de l’expression d’Egr-1 dans les CMLV demeure inabordée; plus spécifiquement, la caractérisation de la voie de signalisation reliant l’action d’IGF-1 à l’expression d’Egr-1 reste à établir. Dans cette optique, l’objectif de cette étude a été d’examiner l’implication de MAPK, PKB et des dérivés réactifs de l’oxygène (DRO) dans l’expression d’Egr-1 induite par l’IGF-1 dans les CMLV. L’IGF-1 a induit une augmentation marquée du niveau protéique de l’Egr-1 en fonction du temps et de la concentration utilisés. Cette augmentation a été inhibée en fonction des doses d’agents pharmacologiques qui ciblent les voies de signalisation de MAPK, PKB et DRO. De plus, l’expression du facteur de transcription, Egr-1, en réponse de l’IGF-1, a été atténuée suite à un blocage pharmacologique des processus cellulaires responsables de la synthèse d’ARN et de synthèse protéique. Pour conclure, on a démontré que l’IGF-1 stimule l’expression d’Egr-1 via les voies de signalisation, impliquant ERK1/2/JNK, PI3K/PKB. On a également proposé que les DRO jouent un rôle important dans ce processus. Dans l’ensemble, nous avons suggéré un nouveau mécanisme par lequel l’IGF-1 promeut la prolifération et l’hypertrophie cellulaire, processus à la base des anomalies vasculaires. / Aberrant vascular remodelling underlies the pathogenesis of major cardiovascular diseases (CVD), such as atherosclerosis and hypertension. Abnormal growth, migration and proliferation of vascular smooth muscle cells (VSMC) are believed to play a critical role in vascular remodelling. IGF-1, potent mitogen, is believed to contribute to the development of CVD through the hyperactivation of proliferative and growth promoting pathways including mitogen-activated protein kinase (MAPK) and protein kinase B (PKB) pathways. It has also been implicated in modulating the expression of multiple transcription factors, including the early growth response protein-1 (Egr-1). Egr-1 upregulation has been observed in different models of vascular diseases implicating growth and redox signalling such as observed in response to IGF-1. However, modulation of Egr-1 expression by IGF-1 in VSMC, more specifically the signaling pathways involved in this process remain poorly characterized. Therefore, in the present studies, we investigated the implication of MAPK, PKB and ROS in IGF-1-induce Egr-1 expression in VSMC. IGF-1 induced a marked increase in Egr-1 protein level in a time and dose-dependent fashion. This increase was dose dependently inhibited by different pharmacological inhibitors targeting MAPK, PKB and reactive oxygen species (ROS) generation. Furthermore, pharmacological inhibitors of RNA and protein synthesis also attenuated IGF-1-induce response on Egr-1 expression. In conclusion, we showed that IGF-1 stimulates the expression of Egr-1 through ERK1/2/JNK and PI3K/PKB. We also propose that ROS generation plays an important role in this response. Overall, we propose a novel mechanism through which IGF-1 may exert its deleterious responses in vascular abnormalities.

O objetivo desse estudo foi avaliar o efeito do tratamento com a aldosterona (Aldo), por 21 dias, sobre a função e a morfologia renal de ratos, procurando correlacionar as alterações com a expressão do EGFR e da ERK1/2 fosforilados. A Aldo não alterou os parâmetros fisiológicos, a PA e a função renal dos ratos, mas verificou-se uma tendência para o aumento das concentrações plasmáticas de K+. No córtex e na medula renal, a Aldo aumentou a expressão do EGFR e da ERK1/2 fosforilados, sendo que este efeito foi abolido pelo tratamento concomitante com a espironolactona ou o RU 486 (antagonistas dos receptores da Aldo). O tratamento hormonal também induziu um aumento na marcação imuno-histoquímica para essas proteínas no córtex e medula; contudo, este efeito foi reduzido, principalmente, com o tratamento conjunto com a espironolactona. Observou-se um aumento na área glomerular e na porcentagem da área intersticial, após o tratamento com a Aldo. As alterações na morfometria renal foram parcialmente reduzidas pelo tratamento com a espironolactona ou o RU 486. Esses resultados indicam que a Aldo pode induzir alterações morfológicas no rim, aparentemente, de maneira dependente dos receptores MR e GR e da via do EGFR-ERK1/2, mas sem o comprometimento da função renal. / The purpose of this study was to evaluate the effect of 21 days of treatment with aldosterone (Aldo) in the renal function and morphology of rats, correlating changes with the expression of EGFR and ERK1/2 phosphorylated. The Aldo did not alter physiological parameters, blood pressure and renal function of rats, but there was a tendency to increased K+ plasma concentrations. In renal cortex and medulla, Aldo increased the expression of EGFR and ERK1/2 phosphorylated and this effect was abolished by treatment Aldo plus spironolactone or RU 486 (Aldo receptor antagonists). Hormonal treatment also induced an increase in immunohistochemical staining for these proteins in the cortex and medulla; however, this effect was limited mainly to treatment Aldo plus spironolactone. There was an increase in glomerular area and the percentage of interstitial area after treatment with Aldo. Changes in renal morphology were partially reduced by treatment Aldo plus spironolactone or RU 486. These results indicate that Aldo may induce morphological changes in the kidney of dependent manner of MR and GR receptors and EGFR-ERK1/2 signaling, but without the impairment of renal function.

No description available.

Signaling scaffolds are critical for the correct spatial organization of enzymes within the ERK1/2 signaling pathway and proper transmission of intracellular information. However, mechanisms that control molecular dynamics within scaffolding complexes, as well as biological activities regulated by the specific assemblies, remain unclear. The scaffold protein Shoc2 is critical for transmission of the ERK1/2 pathway signals. Shoc2 accelerates ERK1/2 signaling by integrating Ras and RAF-1 enzymes into a multi-protein complex. Germ-line mutations in shoc2 cause Noonan-like RASopathy, a disorder with a wide spectrum of developmental deficiencies. However, the physiological role of Shoc2, the nature of ERK1/2 signals transduced through this complex or mechanisms regulating the function of Shoc2 remain largely unknown. My dissertation addresses the mechanisms by which Shoc2 accelerates ERK1/2 signal transmission and the biological outputs of the Shoc2-guided signals. To delineate Shoc2-mediated ERK1/2 signals, I have utilized a vertebrate zebrafish model. I demonstrated that loss of Shoc2 protein expression leads to early embryonic lethality resulting from a significant reduction in the number of circulating erythropoietic and myelopoietic blood cells, underdeveloped neurocranial and pharyngeal cartilages, and a profound delay in calcification of bone structures. Together, this data demonstrates that the Shoc2 scaffolding module transmits ERK1/2 signals in neural crest development and blood cell differentiation. This dissertation also addresses the mechanistic basis of how allosteric ubiquitination of Shoc2 and RAF-1 is controlled. I have characterized a molecular interaction of Shoc2 with its previously unknown binding partner Valosin-Containing Protein (VCP/p97). These studies demonstrated that hexametric ATPase VCP modulates ubiquitination of Shoc2 and RAF-1 through the remodeling of the scaffolding complex in a spatial-restricted manner. Experiments utilizing fluorescence microscopy and biochemical methods show that VCP/p97 sequesters the E3 ligase HUWE1 from the Shoc2 module, thereby altering the ubiquitination of Shoc2 and RAF-1 as well as the amplitude of ERK1/2 signals. These studies also show that the levels of Shoc2 ubiquitination and ERK1/2 phosphorylation are imbalanced in fibroblasts isolated from Inclusion Body Myopathy with Paget’s disease of bone and Frontotemporal Dementia (IBMPFD) patients harboring VCP germline mutations. This data also suggests that ERK1/2 pathway deregulation is part of IBMPFD pathogenesis. In summary, these studies make a significant advance in our understanding of the mechanisms by which the Shoc2 scaffold regulates specificity and the dynamics of the ERK1/2 signaling networks. They also make important insights into our understanding of biological activities and targets of Shoc2-mediated ERK1/2 signals at the early stages of embryonic development and disease.

<p>Parathyroid hormone (PTH) is the major physiological regulator of the extracellular Ca²⁺ concentration ([Ca²⁺]_o) in the body. The secretion of this hormone is suppressed at high [Ca²⁺]_o. Previously this was thought to occur by intracellular degradation of the hormone in the secretory pathway of parathyroid (PT) cells but is now believed to result from extracellular Ca²⁺ stimulus-secretion coupling via the calcium sensing receptor (CaR). In contrast to the stimulation of PTH secretion upon inhibition of mature PTH proteolysis, inhibition of PT proteasomes caused the accumulation of PTH precursors and inhibited secretion of PTH. This suggests that PT proteasomes play a quality control function in the maturation of PTH but they do not directly participate in the [Ca²⁺]_o-regulated secretion of the hormone. Treatment of PT cells with 12-O-tetradecanyolphorbol-13-acetate (TPA) blocks the high [Ca²⁺]_o-induced CaR-mediated suppression of PTH secretion. To delineate the role of DAG-responsive protein kinase C (PKC) isoforms in this process, we complemented pharmacological modulation of PKC activity with physiological activation of the enzyme via the CaR. PKC-α was rapidly activated by high [Ca²⁺]_o and was efficiently down-regulated by prolonged TPA treatment. In CaR-transfected HEK293 cells, TPA and high [Ca²⁺]_o induced the activation of ERK1/2 but the TPA effect was CaR- and Ca²⁺-independent. The magnitude of neomycin-induced release of Ca²⁺ from intracellular stores following pharmacological modulation of PKC activity was opposite to that resulting from physiological activation/inhibition of the enzyme via the CaR. Influx of Ca²⁺ following activation of the receptor occurred by store-operated mechanisms. Over-expression of wt or DN PKC-α or-ε in PT cells using the Tet-On adenovirus gene delivery system revealed that the stimulatory effect of TPA on PTH secretion at high [Ca²⁺]_o was enhanced in cells over-expressing wt PKC-α, but the coupling of the extracellular Ca²⁺ signal to PTH secretion was not dependent on the physiological activation of this PKC isoform via the CaR.</p>

Parathyroid hormone (PTH) is the major physiological regulator of the extracellular Ca2+ concentration ([Ca2+]o) in the body. The secretion of this hormone is suppressed at high [Ca2+]o. Previously this was thought to occur by intracellular degradation of the hormone in the secretory pathway of parathyroid (PT) cells but is now believed to result from extracellular Ca2+ stimulus-secretion coupling via the calcium sensing receptor (CaR). In contrast to the stimulation of PTH secretion upon inhibition of mature PTH proteolysis, inhibition of PT proteasomes caused the accumulation of PTH precursors and inhibited secretion of PTH. This suggests that PT proteasomes play a quality control function in the maturation of PTH but they do not directly participate in the [Ca2+]o-regulated secretion of the hormone. Treatment of PT cells with 12-O-tetradecanyolphorbol-13-acetate (TPA) blocks the high [Ca2+]o-induced CaR-mediated suppression of PTH secretion. To delineate the role of DAG-responsive protein kinase C (PKC) isoforms in this process, we complemented pharmacological modulation of PKC activity with physiological activation of the enzyme via the CaR. PKC-α was rapidly activated by high [Ca2+]o and was efficiently down-regulated by prolonged TPA treatment. In CaR-transfected HEK293 cells, TPA and high [Ca2+]o induced the activation of ERK1/2 but the TPA effect was CaR- and Ca2+-independent. The magnitude of neomycin-induced release of Ca2+ from intracellular stores following pharmacological modulation of PKC activity was opposite to that resulting from physiological activation/inhibition of the enzyme via the CaR. Influx of Ca2+ following activation of the receptor occurred by store-operated mechanisms. Over-expression of wt or DN PKC-α or-ε in PT cells using the Tet-On adenovirus gene delivery system revealed that the stimulatory effect of TPA on PTH secretion at high [Ca2+]o was enhanced in cells over-expressing wt PKC-α, but the coupling of the extracellular Ca2+ signal to PTH secretion was not dependent on the physiological activation of this PKC isoform via the CaR.