Initiation and maintenance of mitosis require the activation of protein kinase cyclin B-Cdc2 and the inhibition of protein phosphatase 2A (PP2A), which, respectively, phosphorylate and dephosphorylate mitotic substrates. The protein kinase Greatwall (Gwl) is required to maintain mitosis through PP2A inhibition. We describe how Gwl activation results in PP2A inhibition. We identified cyclic adenosine monophosphate-regulated phosphoprotein 19 (Arpp19) and α-Endosulfine as two substrates of Gwl that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. Conversely, in the absence of Gwl activity, Arpp19 and α-Endosulfine are dephosphorylated and lose their capacity to bind and inhibit PP2A. Although both proteins can inhibit PP2A, endogenous Arpp19, but not α-Endosulfine, is responsible for PP2A inhibition at mitotic entry in Xenopus egg extracts.
Abstract Hypoxia, IL-1β production and oxidative stress are involved in islet graft dysfunction and destruction. However, the link between these events has not yet been determined in transplanted islets. The goal of this study was to determine whether NLRP3 inflammasome is responsible for IL-1β production and if it is activated by hypoxia-induced oxidative stress in transplanted islets. Rat islets were transplanted under the kidney capsule of immunodeficient mice. At different times post-transplantation, blood samples were collected and islet grafts harvested. Rat islets were also incubated in vitro either under normoxia or hypoxia for 24 h, in the absence or presence of inhibitors of NLRP3 inflammasome (CASP1 inhibitor) or oxidative stress (NAC). NLRP3, CASP1 , IL1B , BBC3 pro-apoptotic and BCL2 anti-apoptotic genes in transplanted and in vitro incubated islets were then studied using real time PCR. IL-1β released in the blood and in the supernatant was quantified by ELISA. Cell death was analysed by propidium iodide and Annexin-V staining. NLRP3 , CASP1 and BBC3 in transplanted rat islets and IL-1β in blood transiently increased during the first days after transplantation. In islets incubated under hypoxia, NRLP3, IL1B and CASP1 and IL-1β released in supernatant increased compared to islets incubated under normoxia. These effects were prevented by the inhibition of NLRP3 inflammasome by CASP1 or oxidative stress by NAC. However, these inhibitors did not prevent hypoxia-induced rat islet death. These data show that NLRP3 inflammasome in rat islets is transiently activated after their transplantation and induced through oxidative stress in vitro . However, NRLP3 inflammasome inhibition does not protect islet cells against hypoxia.
Syndecans (SDC) are important multifunctional components of the extracellular matrix mainly described in endothelial cells. We studied the expression and regulation of SDC in cultured MIN6B1 cells and pancreatic islets. qRT-PCR revealed that syndecan-4 (SDC4) was the predominant isoform expressed in MIN6B1 cells and islets compared to other forms of SDC. Immunofluorescence in mouse and human pancreas sections revealed that SDC4 is mainly expressed in β-cells compared to other pancreatic cells. Exposure of MIN6B1 and human islets to IL-1β dose-dependently induced a rapid and transient expression of SDC4 while SRC and STAT3 inhibitors decreased this effect. Exposure of human islets to Il-1β caused an increase of SDC4 shedding, however treatment with STAT3 and SRC inhibitors inhibited this effect. These results indicate that SDC4 is upregulated by IL-1β through the SRC-STAT3 pathway and this pathway is also involved in SDC4 shedding in islets.
The aim of this study was to evaluate the location of PP and δ cells in relation to the vascularization within human pancreatic islets. To this end, pancreas sections were analysed by immunofluorescence using antibodies against endocrine islet and endothelial cells. Staining in different islet areas corresponding to islet cells adjacent or not to peripheral or central vascular channels was quantified by computerized morphometry. As results, α, PP and δ cells were preferentially found adjacent to vessels. In contrast to α cells, which were evenly distributed between islet periphery and intraislet vascular channels, PP and δ cells had asymmetric and opposite distributions: PP staining was higher and somatostatin staining was lower in the islet periphery than in the area around intraislet vascular channels. Additionally, frequencies of PP and δ cells were negatively correlated in the islets. No difference was observed between islets from the head and the tail of the pancreas, and from type 2 diabetic and non-diabetic donors. In conclusion, the distribution of δ cells differs from that of PP cells in human islets, suggesting that vessels at the periphery and at the centre of islets drain different hormonal cocktails.
Abstract TLR4 is a transmembrane receptor of the innate immune system that recognizes LPS from gram-negative bacteria. Its stimulation induces pro-inflammatory responses and modulates adaptive immunity. Our aim is to determine the role of TLR4 in the activation and proliferation of T lymphocytes in the onset of autoimmune diabetes, using the non-obese diabetic (NOD) mouse model. Antigen-specific activation and proliferation of diabetogenic T cells were assessed in vitro by Carboxyfluorescein succinimidyl ester (CFSE) dilution, in presence of vehicle or CLI-095, a cyclohexene derivative that inhibits TLR4 signaling. NOD mice were treated with vehicle or CLI-095 and sacrificed either before or after the onset of autoimmune diabetes. T lymphocyte activation and proliferation were evaluated in treated and control mice. Insulitis was analyzed by histology and diabetes incidence was determined in treated and control mice. Our results demonstrate that TLR4 blockade decreases CD4+ T lymphocyte activation and auto-antigen-specific proliferation both in vitro and in vivo , decreases the infiltrative insulitis and finally prevents the onset of spontaneous diabetes. Taken together, our data demonstrate that TLR4 signaling contributes to the development and maintenance of autoimmune diabetes. The immunomodulatory effect of CLI-095 could be part of a preventive strategy targeting patients at risk for type 1 diabetes.
La mitose est une phase importante du cycle cellulaire. Les mecanismes de surveillance s'assurent de l'ordre et de l'execution correcte des evenements du cycle cellulaire dont les erreurs peuvent conduire a l'aneuploidie. Pendant la mitose, la separation des chromatides sœurs est regulee par le point de controle du fuseau mitotique qui s'assure que tous les chromosomes sont correctement alignes sur la plaque metaphasique. L'entree et la sortie de mitose sont regulees par l'activation et l'inactivation du complexe cycline B/Cdk1. Cette fine regulation fait intervenir de nombreuses kinases et phosphatases. Dans ce projet nous nous sommes interesses plus particulierement a deux AGC kinases : RSK2 et Greatwall (Gwl).Au cours de cette etude nous nous sommes proposes d'analyser l'implication de RSK2, substrat majeur de la MAPK, dans le point de controle du fuseau mitotique. Nos resultats montrent que RSK2 est essentielle pour l'activite du point de controle du fuseau mitotique dans les extraits d'œufs de xenope ainsi que pour la localisation des autres proteines de ce mecanisme de surveillance localisees aux kinetochores. Nous montrons egalement que RSK2 participe au point de controle dans les cellules humaines. En effet, RSK2 est necessaire a la localisation aux kinetochores de Mad1, Mad2 et Cenp-E, proteines essentielles a l'activite de ce checkpoint. L'entree et la sortie de mitose sont regulees par le complexe cycline B/Cdk1 et des phosphatases. Gwl est une nouvelle kinase essentielle a l'entree en mitose et au maintien de l'etat mitotique dans les extraits d'œufs de xenope. En effet, nos resultats montrent que Gwl maintient l'etat mitotique independamment du complexe cycline B/Cdk1, en regulant negativement PP2A, une phosphatase responsable de la dephoshorylation des substrats mitotiques.
The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted to vesicular membrane pathways and synaptic clusters in neurons by both a GAD65-dependent and a distinct GAD65-independent mechanism. Herein we have investigated the membrane association and targeting of GAD67 and GAD65 in monolayer cultures of primary rat, human, and mouse islets and in insulinoma cells. GAD65 is primarily detected in Golgi membranes and in peripheral vesicles distinct from insulin vesicles in β-cells. In the absence of GAD65, GAD67 is in contrast primarily cytosolic in β-cells; its co-expression with GAD65 is necessary for targeting to Golgi membranes and vesicular compartments. Thus, the GAD65-independent mechanism for targeting of GAD67 to synaptic vesicles in neurons is not functional in islet β-cells. Therefore, only GAD65:GAD65 homodimers and GAD67:GAD65 heterodimers, but not the GAD67:GAD67 homodimer gain access to vesicular compartments in β-cells to facilitate rapid accumulation of newly synthesized GABA for regulated secretion and fine tuning of GABA-signaling in islets of Langerhans.
Here we show that the functional human ortholog of Greatwall protein kinase (Gwl) is the microtubule-associated serine/threonine kinase-like protein, MAST-L. This kinase promotes mitotic entry and maintenance in human cells by inhibiting protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates cyclin B-Cdc2 substrates. The complete depletion of Gwl by siRNA arrests human cells in G2. When the levels of this kinase are only partially depleted, however, cells enter into mitosis with multiple defects and fail to inactivate the spindle assembly checkpoint (SAC). The ability of cells to remain arrested in mitosis by the SAC appears to be directly proportional to the amount of Gwl remaining. Thus, when Gwl is only slightly reduced, cells arrest at prometaphase. More complete depletion correlates with the premature dephosphorylation of cyclin B-Cdc2 substrates, inactivation of the SAC, and subsequent exit from mitosis with severe cytokinesis defects. These phenotypes appear to be mediated by PP2A, as they could be rescued by either a double Gwl/PP2A knockdown or by the inhibition of this phos-phatase with okadaic acid. These results suggest that the balance between cyclin B-Cdc2 and PP2A must be tightly regulated for correct mitotic entry and exit and that Gwl is crucial for mediating this regulation in somatic human cells.
