8 research outputs found
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Rho Family GTPases and Rho GEFs in Glucose Homeostasis.
Dysregulation of glucose homeostasis leading to metabolic syndrome and type 2 diabetes is the cause of an increasing world health crisis. New intriguing roles have emerged for Rho family GTPases and their Rho guanine nucleotide exchange factor (GEF) activators in the regulation of glucose homeostasis. This review summates the current knowledge, focusing in particular on the roles of Rho GEFs in the processes of glucose-stimulated insulin secretion by pancreatic β cells and insulin-stimulated glucose uptake into skeletal muscle and adipose tissues. We discuss the ten Rho GEFs that are known so far to regulate glucose homeostasis, nine of which are in mammals, and one is in yeast. Among the mammalian Rho GEFs, P-Rex1, Vav2, Vav3, Tiam1, Kalirin and Plekhg4 were shown to mediate the insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane and/or insulin-stimulated glucose uptake in skeletal muscle or adipose tissue. The Rho GEFs P-Rex1, Vav2, Tiam1 and β-PIX were found to control the glucose-stimulated release of insulin by pancreatic β cells. In vivo studies demonstrated the involvement of the Rho GEFs P-Rex2, Vav2, Vav3 and PDZ-RhoGEF in glucose tolerance and/or insulin sensitivity, with deletion of these GEFs either contributing to the development of metabolic syndrome or protecting from it. This research is in its infancy. Considering that over 80 Rho GEFs exist, it is likely that future research will identify more roles for Rho GEFs in glucose homeostasis
Dock2 generates characteristic spatiotemporal patterns of Rac activity to regulate neutrophil polarisation, migration and phagocytosis
IntroductionRac-GTPases and their Rac-GEF activators play important roles in neutrophil-mediated host defence. These proteins control the adhesion molecules and cytoskeletal dynamics required for neutrophil recruitment to inflamed and infected organs, and the neutrophil effector responses that kill pathogens.MethodsHere, we used live cell TIRF-FRET imaging in neutrophils from Rac-FRET reporter mice with deficiencies in the Rac-GEFs Dock2, Tiam1 or Prex1/Vav1 to evaluate if these proteins activate spatiotemporally distinct pools of Rac, and to correlate patterns of Rac activity with the neutrophil responses they control.ResultsAll the GEFs were required for neutrophil adhesion, and Prex1/Vav1 were important during spreading and for the velocity of migration during chemotaxis. However, Dock2 emerged as the prominent regulator of neutrophil responses, as this GEF was required for neutrophil polarisation and random migration, for migration velocity during chemokinesis, for the likelihood to migrate and for the speed of migration and of turning during chemotaxis, as well as for rapid particle engulfment during phagocytosis. We identified characteristic spatiotemporal patterns of Rac activity generated by Dock2 which correlate with the importance of the Rac-GEF in these neutrophil responses. We also demonstrate a requirement for Dock2 in neutrophil recruitment during aseptic peritonitis.DiscussionCollectively, our data provide a first direct comparison of the pools of Rac activity generated by different types of Rac-GEFs, and identify Dock2 as a key regulator of polarisation, migration and phagocytosis in primary neutrophils
The Rac-GEF Tiam1 controls integrin-dependent neutrophil responses
Rac GTPases are required for neutrophil adhesion and migration, and for the neutrophil effector responses that kill pathogens. These Rac-dependent functions are impaired when neutrophils lack the activators of Rac, Rac-GEFs from the Prex, Vav, and Dock families. In this study, we demonstrate that Tiam1 is also expressed in neutrophils, governing focal complexes, actin cytoskeletal dynamics, polarisation, and migration, in a manner depending on the integrin ligand to which the cells adhere. Tiam1 is dispensable for the generation of reactive oxygen species but mediates degranulation and NETs release in adherent neutrophils, as well as the killing of bacteria. In vivo, Tiam1 is required for neutrophil recruitment during aseptic peritonitis and for the clearance of Streptococcus pneumoniae during pulmonary infection. However, Tiam1 functions differently to other Rac-GEFs. Instead of promoting neutrophil adhesion to ICAM1 and stimulating β2 integrin activity as could be expected, Tiam1 restricts these processes. In accordance with these paradoxical inhibitory roles, Tiam1 limits the fMLP-stimulated activation of Rac1 and Rac2 in adherent neutrophils, rather than activating Rac as expected. Tiam1 promotes the expression of several regulators of small GTPases and cytoskeletal dynamics, including αPix, Psd4, Rasa3, and Tiam2. It also controls the association of Rasa3, and potentially αPix, Git2, Psd4, and 14-3-3ζ/δ, with Rac. We propose these latter roles of Tiam1 underlie its effects on Rac and β2 integrin activity and on cell responses. Hence, Tiam1 is a novel regulator of Rac-dependent neutrophil responses that functions differently to other known neutrophil Rac-GEFs
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New roles of Rac-GEFs in Neutrophils and in Glucose Homeostasis
Rac-GEFs (guanine-nucleotide exchange factors) are proteins that activate Rac GTPases, thereby enabling Rac-dependent cytoskeletal dynamics and cellular processes such as adhesion and migration. I used mice with genetically modified Rac-GEFs to identify new functional roles of these proteins in two distinct biological systems, neutrophil adhesion/migration and glucose homeostasis.
In the first part of my thesis, I investigated cytoskeletal dynamics controlled by the Rac-GEF Tiam1 in neutrophil adhesion/migration. We previously found a paradoxical increase in the adhesion of Tiam1–/– neutrophils (unpublished). This was surprising, as deficiencies in other neutrophil Rac-GEFs impair adhesion. I showed deregulated neutrophil polarisation, Filamentous-actin (F-actin) polarity, F-actin dynamics and focal adhesion structures in Tiam1–/– neutrophils adhering to integrin ligands. I demonstrated increased integrin avidity in Tiam1–/– neutrophils stimulated with CXCL1, and increased migration of Tiam1–/– neutrophils under shear stress. These results contribute to our elucidation of the mechanisms underlying the paradoxical increase in the adhesion of Tiam1-deficient neutrophils.
In the second part, I investigated spatiotemporal patterns of Rac activity generated during neutrophil adhesion/migration by several major neutrophil Rac-GEFs. The aim was to identify specific roles for these Rac-GEFs which all signal in response to the activation of GPCRs. I used our Rac activity FRET reporter mouse strain (RFC) (Johnsson, Dai et al. 2014), crossed to mice deficient in the Rac-GEFs P-Rex1/Vav1, DOCK2 or Tiam1. I demonstrated that Rac activity is increased in RFC Tiam1–/– neutrophils adhering to integrin ligands, which may explain the increased adhesion. In contrast, RFC DOCK2–/– and RFC P-Rex1–/– Vav1–/– neutrophils had reduced Rac activity, as expected for Rac-GEF deficient cells, confirming a unique and paradoxical role of Tiam1 in limiting Rac activity and Rac-dependent cell responses. The loss of Rac activity was global in RFC DOCK2–/– neutrophils but more localised in RFC P-Rex1–/– Vav1–/– cells. This project has identified specific roles of various Rac-GEFs in neutrophil adhesion and migration.
In the third and final part, I investigated adaptor functions of P-Rex family Rac-GEFs P-Rex1 and P-Rex2 in glucose homeostasis. We previously showed that P-Rex1 and P-Rex2 deficient mice have accelerated glucose clearance during glucose challenge, along with low fasting blood glucose levels and altered insulin sensitivity (unpublished). In order to address the underlying mechanisms, I used mice with catalytically inactive P-Rex1 or P-Rex2 (GEF-dead mice) which we recently generated (unpublished). I demonstrated that the increased glucose clearance is an adaptor function of P-Rex Rac-GEFs, whereas fasting blood glucose levels and insulin sensitivity are Rac-GEF activity dependent. I showed increased plasma insulin levels in P-Rex1–/– and P-Rex2–/– mice upon glucose challenge and increased glucose-stimulated insulin secretion from P-Rex1–/– and P-Rex2–/– pancreatic islets. Use of P-Rex1 GEF-dead mice showed that these latter phenotypes were again adaptor functions, suggesting that these responses contribute to the accelerated glucose clearance in P-Rex-deficient mice.
Combined, my work has provided a substantial body of data identifying unexpected novel roles for Rac-GEFs in both neutrophil biology and in glucose homeostasis, providing mechanistic insight in addition to new functions in both systems
Rho Family GTPases and Rho GEFs in Glucose Homeostasis
Dysregulation of glucose homeostasis leading to metabolic syndrome and type 2 diabetes is the cause of an increasing world health crisis. New intriguing roles have emerged for Rho family GTPases and their Rho guanine nucleotide exchange factor (GEF) activators in the regulation of glucose homeostasis. This review summates the current knowledge, focusing in particular on the roles of Rho GEFs in the processes of glucose-stimulated insulin secretion by pancreatic β cells and insulin-stimulated glucose uptake into skeletal muscle and adipose tissues. We discuss the ten Rho GEFs that are known so far to regulate glucose homeostasis, nine of which are in mammals, and one is in yeast. Among the mammalian Rho GEFs, P-Rex1, Vav2, Vav3, Tiam1, Kalirin and Plekhg4 were shown to mediate the insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane and/or insulin-stimulated glucose uptake in skeletal muscle or adipose tissue. The Rho GEFs P-Rex1, Vav2, Tiam1 and β-PIX were found to control the glucose-stimulated release of insulin by pancreatic β cells. In vivo studies demonstrated the involvement of the Rho GEFs P-Rex2, Vav2, Vav3 and PDZ-RhoGEF in glucose tolerance and/or insulin sensitivity, with deletion of these GEFs either contributing to the development of metabolic syndrome or protecting from it. This research is in its infancy. Considering that over 80 Rho GEFs exist, it is likely that future research will identify more roles for Rho GEFs in glucose homeostasis
The GPCR adaptor protein norbin suppresses the neutrophil-mediated immunity of mice to pneumococcal infection
Streptococcal pneumonia is a worldwide health problem that kills 2 million people each year, particularly young children, the elderly, and immunosuppressed individuals. Alveolar macrophages and neutrophils provide the early innate immune response to clear pneumococcus from infected lungs. However, the level of neutrophil involvement is context dependent, both in humans and in mouse models of the disease, influenced by factors such as bacterial load, age, and coinfections. Here, we show that the G protein coupled receptor (GPCR) adaptor protein norbin (neurochondrin, NCDN), which was hitherto known as a regulator of neuronal function, is a suppressor of neutrophilmediated innate immunity. Myeloid norbin deficiency improved the immunity of mice to pneumococcal infection by increasing the involvement of neutrophils in clearing the bacteria, without affecting neutrophil recruitment or causing autoinflammation. It also improved immunity during Escherichia coli induced septic peritonitis. It increased the responsiveness of neutrophils to a range of stimuli, promoting their ability to kill bacteria in a reactive oxygen species dependent manner, enhancing degranulation, phagocytosis, and the production of reactive oxygen species and neutrophil extracellular traps, raising the cell surface levels of selected GPCRs, and increasing GPCR-dependent Rac and Erk signaling. The Rac guanine-nucleotide exchange factor Prex1, a known effector of norbin, was dispensable for most of these effects, which suggested that norbin controls additional downstream targets. We identified the Rac guanine-nucleotide exchange factor Vav as one of these effectors. In summary, our study presents the GPCR adaptor protein norbin as an immune suppressor that limits the ability of neutrophils to clear bacterial infections