SHP-1 phosphatase activity counteracts increased T cell receptor affinity.

Anti-self/tumor T cell function can be improved by increasing TCR-peptide MHC (pMHC) affinity within physiological limits, but paradoxically further increases (K(d) < 1 μM) lead to drastic functional declines. Using human CD8(+) T cells engineered with TCRs of incremental affinity for the tumor antigen HLA-A2/NY-ESO-1, we investigated the molecular mechanisms underlying this high-affinity-associated loss of function. As compared with cells expressing TCR affinities generating optimal function (K(d) = 5 to 1 μM), those with supraphysiological affinity (K(d) = 1 μM to 15 nM) showed impaired gene expression, signaling, and surface expression of activatory/costimulatory receptors. Preferential expression of the inhibitory receptor programmed cell death-1 (PD-1) was limited to T cells with the highest TCR affinity, correlating with full functional recovery upon PD-1 ligand 1 (PD-L1) blockade. In contrast, upregulation of the Src homology 2 domain-containing phosphatase 1 (SHP-1/PTPN6) was broad, with gradually enhanced expression in CD8(+) T cells with increasing TCR affinities. Consequently, pharmacological inhibition of SHP-1 with sodium stibogluconate augmented the function of all engineered T cells, and this correlated with the TCR affinity-dependent levels of SHP-1. These data highlight an unexpected and global role of SHP-1 in regulating CD8(+) T cell activation and responsiveness and support the development of therapies inhibiting protein tyrosine phosphatases to enhance T cell-mediated immunity.

Early diabetes and abnormal postnatal pancreatic islet development in mice lacking Glut-2.

Glut-2 is a low-affinity transporter present in the plasma membrane of pancreatic beta-cells, hepatocytes and intestine and kidney absorptive epithelial cells of mice. In beta-cells, Glut-2 has been proposed to be active in the control of glucose-stimulated insulin secretion (GSIS; ref. 2), and its expression is strongly reduced in glucose-unresponsive islets from different animal models of diabetes. However, recent investigations have yielded conflicting data on the possible role of Glut-2 in GSIS. Whereas some reports have supported a specific role for Glut-2 (refs 5,6), others have suggested that GSIS could proceed normally even in the presence of low or almost undetectable levels of this transporter. Here we show that homozygous, but not heterozygous, mice deficient in Glut-2 are hyperglycaemic and relatively hypo-insulinaemic and have elevated plasma levels of glucagon, free fatty acids and beta-hydroxybutyrate. In vivo, their glucose tolerance is abnormal. In vitro, beta-cells display loss of control of insulin gene expression by glucose and impaired GSIS with a loss of first phase but preserved second phase of secretion, while the secretory response to non-glucidic nutrients or to D-glyceraldehyde is normal. This is accompanied by alterations in the postnatal development of pancreatic islets, evidenced by an inversion of the alpha- to beta-cell ratio. Glut-2 is thus required to maintain normal glucose homeostasis and normal function and development of the endocrine pancreas. Its absence leads to symptoms characteristic of non-insulin-dependent diabetes mellitus.

RasGAP-derived fragment N increases the resistance of beta cells towards apoptosis in NOD mice and delays the progression from mild to overt diabetes.

The caspase-3-generated RasGAP N-terminal fragment (fragment N) inhibits apoptosis in a Ras-PI3K-Akt-dependent manner. Fragment N protects various cell types, including insulin-secreting cells, against different types of stresses. Whether fragment N exerts a protective role during the development of type 1 diabetes is however not known. Non-obese diabetic (NOD) mice represent a well-known model for spontaneous development of type 1 diabetes that shares similarities with the diseases encountered in humans. To assess the role of fragment N in type 1 diabetes development, a transgene encoding fragment N under the control of the rat insulin promoter (RIP) was back-crossed into the NOD background creating the NOD-RIPN strain. Despite a mosaic expression of fragment N in the beta cell population of NOD-RIPN mice, islets isolated from these mice were more resistant to apoptosis than control NOD islets. Islet lymphocytic infiltration and occurrence of a mild increase in glycemia developed with the same kinetics in both strains. However, the period of time separating the mild increase in glycemia and overt diabetes was significantly longer in NOD-RIPN mice compared to the control NOD mice. There was also a significant decrease in the number of apoptotic beta cells in situ at 16 weeks of age in the NOD-RIPN mice. Fragment N exerts therefore a protective effect on beta cells within the pro-diabetogenic NOD background and this prevents a fast progression from mild to overt diabetes.

Molecular pathways involved in the antineoplastic effects of calcitriol on insulinoma cells.

We have previously reported that in tumorigenic pancreatic beta-cells, calcitriol exerts a potent antitumorigenic effect by inducing apoptosis, cell growth inhibition, and reduction of solid beta-cell tumors. Here we have studied the molecular pathways involved in the antineoplastic activity of calcitriol on mouse insulinoma beta TC(3) cells, mouse insulinoma beta TC expressing or not expressing the oncogene p53, and beta TC-tet cells overexpressing or not the antiapoptotic gene Bcl2. Our results indicate that calcitriol-induced apoptosis was dependent on the function of p53 and was associated with a biphasic increase in protein levels of transcription factor nuclear factor-kappa B. Calcitriol decreased cell viability by about 40% in p53-retaining beta TC and in beta TC(3) cells; in contrast, beta TC p53(-/-) cells were only minimally affected. Calcitriol-induced cell death was regulated by members of the Bcl-2 family of apoptosis regulatory proteins, as shown by calcitriol-induced up-regulation of proapoptotic Bax and Bak and the lack of calcitriol-induced cytotoxicity in Bcl-2-overexpressing insulinoma cells. Moreover, calcitriol-mediated arrest of beta TC(3) cells in the G(1) phase of the cell cycle was associated with the abnormal expression of p21 and G(2)/M-specific cyclin B2 genes and involved the DNA damage-inducible factor GADD45. Finally, in beta TC(3) cells, calcitriol modulated the expression of IGF-I and IGF-II genes. In conclusion, these findings contribute to the understanding of the antitumorigenic effects of calcitriol on tumorigenic pancreatic beta-cells and further support the rationale of its utilization in the treatment of patients with malignant insulinomas.

GLUT2, glucose sensing and glucose homeostasis.

The glucose transporter isoform GLUT2 is expressed in liver, intestine, kidney and pancreatic islet beta cells, as well as in the central nervous system, in neurons, astrocytes and tanycytes. Physiological studies of genetically modified mice have revealed a role for GLUT2 in several regulatory mechanisms. In pancreatic beta cells, GLUT2 is required for glucose-stimulated insulin secretion. In hepatocytes, suppression of GLUT2 expression revealed the existence of an unsuspected glucose output pathway that may depend on a membrane traffic-dependent mechanism. GLUT2 expression is nevertheless required for the physiological control of glucose-sensitive genes, and its inactivation in the liver leads to impaired glucose-stimulated insulin secretion, revealing a liver-beta cell axis, which is likely to be dependent on bile acids controlling beta cell secretion capacity. In the nervous system, GLUT2-dependent glucose sensing controls feeding, thermoregulation and pancreatic islet cell mass and function, as well as sympathetic and parasympathetic activities. Electrophysiological and optogenetic techniques established that Glut2 (also known as Slc2a2)-expressing neurons of the nucleus tractus solitarius can be activated by hypoglycaemia to stimulate glucagon secretion. In humans, inactivating mutations in GLUT2 cause Fanconi-Bickel syndrome, which is characterised by hepatomegaly and kidney disease; defects in insulin secretion are rare in adult patients, but GLUT2 mutations cause transient neonatal diabetes. Genome-wide association studies have reported that GLUT2 variants increase the risks of fasting hyperglycaemia, transition to type 2 diabetes, hypercholesterolaemia and cardiovascular diseases. Individuals with a missense mutation in GLUT2 show preference for sugar-containing foods. We will discuss how studies in mice help interpret the role of GLUT2 in human physiology.

The C76R transmembrane activator and calcium modulator cyclophilin ligand interactor mutation disrupts antibody production and B-cell homeostasis in heterozygous and homozygous mice.

BackgroundMutations in TNFRSF13B, the gene encoding transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), are found in 10% of patients with common variable immunodeficiency. However, the most commonly detected mutation is the heterozygous change C104R, which is also found in 0.5% to 1% of healthy subjects. The contribution of the C104R mutation to the B-cell defects observed in patients with common variable immunodeficiency therefore remains unclear.ObjectiveWe sought to define the functional consequences of the C104R mutation on B-cell function.MethodsWe performed in vitro studies of TACI C104R expression and signaling. A knock-in mouse with the equivalent mutation murine TACI (mTACI) C76R was generated as a physiologically relevant model of human disease. We examined homozygous and heterozygous C76R mutant mice alongside wild-type littermates and studied specific B-cell lineages and antibody responses to T cell-independent and T cell-dependent challenge.ResultsC104R expression and ligand binding are significantly diminished when the mutant protein is expressed in 293T cells or in patients' cell lines. This leads to defective nuclear factor κB activation, which is proportionally restored by reintroduction of wild-type TACI. Mice heterozygous and homozygous for mTACI C76R exhibit significant B-cell dysfunction with splenomegaly, marginal zone B-cell expansion, diminished immunoglobulin production and serological responses to T cell-independent antigen, and abnormal immunoglobulin synthesis.ConclusionsThese data show that the C104R mutation and its murine equivalent, C76R, can significantly disrupt TACI function, probably through haploinsufficiency. Furthermore, the heterozygous C76R mutation alone is sufficient to disturb B-cell function with lymphoproliferation and immunoglobulin production defects.

GLUT2 surface expression and intracellular transport via the constitutive pathway in pancreatic beta cells and insulinoma: evidence for a block in trans-Golgi network exit by brefeldin A.

The biosynthesis, intracellular transport, and surface expression of the beta cell glucose transporter GLUT2 was investigated in isolated islets and insulinoma cells. Using a trypsin sensitivity assay to measure cell surface expression, we determined that: (a) greater than 95% of GLUT2 was expressed on the plasma membrane; (b) GLUT2 did not recycle in intracellular vesicles; and (c) after trypsin treatment, reexpression of the intact transporter occurred with a t1/2 of approximately 7 h. Kinetics of intracellular transport of GLUT2 was investigated in pulse-labeling experiments combined with glycosidase treatment and the trypsin sensitivity assay. We determined that transport from the endoplasmic reticulum to the trans-Golgi network (TGN) occurred with a t1/2 of 15 min and that transport from the TGN to the plasma membrane required a similar half-time. When added at the start of a pulse-labeling experiment, brefeldin A prevented exit of GLUT2 from the endoplasmic reticulum. When the transporter was first accumulated in the TGN during a 15-min period of chase, but not following a low temperature (22 degrees C) incubation, addition of brefeldin A (BFA) prevented subsequent surface expression of the transporter. This indicated that brefeldin A prevented GLUT2 exit from the TGN by acting at a site proximal to the 22 degrees C block. Together, these data demonstrate that GLUT2 surface expression in beta cells is via the constitutive pathway, that transport can be blocked by BFA at two distinct steps and that once on the surface, GLUT2 does not recycle in intracellular vesicles.

Assay for high glucose-mediated islet cell sensitization to apoptosis induced by streptozotocin and cytokines

Pancreatic beta-cell apoptosis is known to participate in the beta-cell destruction process that occurs in diabetes. It has been described that high glucose level induces a hyperfunctional status which could provoke apoptosis. This phenomenon is known as glucotoxicity and has been proposed that it can play a role in type 1 diabetes mellitus pathogenesis. In this study we develop an experimental design to sensitize pancreatic islet cells by high glucose to streptozotocin (STZ) and proinflammatory cytokines [interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma]-induced apoptosis. This method is appropriate for subsequent quantification of apoptotic islet cells stained with Tdt-mediated dUTP Nick-End Labeling (TUNEL) and protein expression assays by Western Blotting (WB).

Role and therapeutic potential of microRNAs in diabetes.

The discovery in mammalian cells of hundreds of small RNA molecules, called microRNAs, with the potential to modulate the expression of the majority of the protein-coding genes has revolutionized many areas of biomedical research, including the diabetes field. MicroRNAs function as translational repressors and are emerging as key regulators of most, if not all, physiological processes. Moreover, alterations in the level or function of microRNAs are associated with an increasing number of diseases. Here, we describe the mechanisms governing the biogenesis and activities of microRNAs. We present evidence for the involvement of microRNAs in diabetes mellitus, by outlining the contribution of these small RNA molecules in the control of pancreatic beta-cell functions and by reviewing recent studies reporting changes in microRNA expression in tissues isolated from diabetes animal models. MicroRNAs hold great potential as therapeutic targets. We describe the strategies developed for the delivery of molecules mimicking or blocking the function of these tiny regulators of gene expression in living animals. In addition, because changes in serum microRNA profiles have been shown to occur in association with different human diseases, we also discuss the potential use of microRNAs as blood biomarkers for prevention and management of diabetes.

The scaffold protein IB1/JIP-1 is a critical mediator of cytokine-induced apoptosis in pancreatic beta cells.

In insulin-secreting cells, cytokines activate the c-Jun N-terminal kinase (JNK), which contributes to a cell signaling towards apoptosis. The JNK activation requires the presence of the murine scaffold protein JNK-interacting protein 1 (JIP-1) or human Islet-brain 1(IB1), which organizes MLK3, MKK7 and JNK for proper signaling specificity. Here, we used adenovirus-mediated gene transfer to modulate IB1/JIP-1 cellular content in order to investigate the contribution of IB1/JIP-1 to beta-cell survival. Exposure of the insulin-producing cell line INS-1 or isolated rat pancreatic islets to cytokines (interferon-gamma, tumor necrosis factor-alpha and interleukin-1beta) induced a marked reduction of IB1/JIP-1 content and a concomitant increase in JNK activity and apoptosis rate. This JNK-induced pro-apoptotic program was prevented in INS-1 cells by overproducing IB1/JIP-1 and this effect was associated with inhibition of caspase-3 cleavage. Conversely, reducing IB1/JIP-1 content in INS-1 cells and isolated pancreatic islets induced a robust increase in basal and cytokine-stimulated apoptosis. In heterozygous mice carrying a selective disruption of the IB1/JIP-1 gene, the reduction in IB1/JIP-1 content in happloinsufficient isolated pancreatic islets was associated with an increased JNK activity and basal apoptosis. These data demonstrate that modulation of the IB1-JIP-1 content in beta cells is a crucial regulator of JNK signaling pathway and of cytokine-induced apoptosis.

Critical role of the transcriptional repressor neuron-restrictive silencer factor in the specific control of connexin36 in insulin-producing cell lines.

Connexin36 (Cx36) is specifically expressed in neurons and in pancreatic beta-cells. Cx36 functions as a critical regulator of insulin secretion and content in beta-cells. In order to identify the molecular mechanisms that control the beta-cell expression of Cx36, we initiated the characterization of the human 5' regulatory region of the CX36 gene. A 2043-bp fragment of the human CX36 promoter was identified from a human BAC library and fused to a luciferase reporter gene. This promoter region was sufficient to confer specific expression to the reporter gene in insulin-secreting cell lines. Within this 5' regulatory region, a putative neuron-restrictive silencer element conserved between rodent and human species was recognized and binds the neuron-restrictive silencing factor (NRSF/REST). This factor is not expressed in insulin-secreting cells and neurons; it functions as a potent repressor through the recruitment of histone deacetylase to the promoter of neuronal genes. The NRSF-mediated repression of Cx36 in HeLa cells was abolished by trichostatin A, confirming the functional importance of histone deacetylase activity. Ectopic expression, by viral gene transfer, of NRSF/REST in different insulin-secreting beta-cell lines induced a marked reduction in Cx36 mRNA and protein content. Moreover, mutations in the Cx36 neuron-restrictive silencer element relieved the low transcriptional activity of the human CX36 promoter observed in HeLa cells and in INS-1 cells expressing NRSF/REST. The data showed that cx36 gene expression in insulin-producing beta-cell lines is strictly controlled by the transcriptional repressor NRSF/REST indicating that Cx36 participates to the neuronal phenotype of the pancreatic beta-cells.

The JNK binding domain of islet-brain 1 inhibits IL-1 induced JNK activity and apoptosis but not the transcription of key proapoptotic or protective genes in insulin-secreting cell lines.

The stress-activated protein kinase c-Jun NH2-terminal kinase (JNK) is a central signal for interleukin-1beta (IL-1beta)-induced apoptosis in insulin-producing beta-cells. The cell-permeable peptide inhibitor of JNK (JNKI1), that introduces the JNK binding domain (JBD) of the scaffold protein islet-brain 1 (IB1) inside cells, effectively prevents beta-cell death caused by this cytokine. To define the molecular targets of JNK involved in cytokine-induced beta-cell apoptosis we investigated whether JNKI1 or stable expression of JBD affected the expression of selected pro- and anti-apoptotic genes induced in rat (RIN-5AH-T2B) and mouse (betaTC3) insulinoma cells exposed to IL-1beta. Inhibition of JNK significantly reduced phosphorylation of the specific JNK substrate c-Jun (p&lt;0.05), IL-1beta-induced apoptosis (p&lt;0.001), and IL-1beta-mediated c-fos gene expression. However, neither JNKI1 nor JBD did influence IL-1beta-induced NO synthesis or iNOS expression or the transcription of the genes encoding mitochondrial manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase rho (GSTrho), heat shock protein (HSP) 70, IL-1beta-converting enzyme (ICE), caspase-3, apoptosis-inducing factor (AIF), Bcl-2 or Bcl-xL. We suggest that the anti-apoptotic effect of JNK inhibition by JBD is independent of the transcription of major pro- and anti-apoptotic genes, but may be exerted at the translational or posttranslational level.

The transcriptional repressor REST determines the cell-specific expression of the human MAPK8IP1 gene encoding IB1 (JIP-1).

Islet-brain 1 (IB1) is the human and rat homologue of JIP-1, a scaffold protein interacting with the c-Jun amino-terminal kinase (JNK). IB1 expression is mostly restricted to the endocrine pancreas and to the central nervous system. Herein, we explored the transcriptional mechanism responsible for this preferential islet and neuronal expression of IB1. A 731-bp fragment of the 5' regulatory region of the human MAPK8IP1 gene was isolated from a human BAC library and cloned upstream of a luciferase reporter gene. This construct drove high transcriptional activity in both insulin-secreting and neuron-like cells but not in unrelated cell lines. Sequence analysis of this promoter region revealed the presence of a neuron-restrictive silencer element (NRSE) known to bind repressor zinc finger protein REST. This factor is not expressed in insulin-secreting and neuron-like cells. By mobility shift assay, we confirmed that REST binds to the NRSE present in the IB1 promoter. Once transiently transfected in beta-cell lines, the expression vector encoding REST repressed IB1 transcriptional activity. The introduction of a mutated NRSE in the 5' regulating region of the IB1 gene abolished the repression activity driven by REST in insulin-secreting beta cells and relieved the low transcriptional activity of IB1 observed in unrelated cells. Moreover, transfection in non-beta and nonneuronal cell lines of an expression vector encoding REST lacking its transcriptional repression domain relieved IB1 promoter activity. Last, the REST-mediated repression of IB1 could be abolished by trichostatin A, indicating that deacetylase activity is required to allow REST repression. Taken together, these data establish a critical role for REST in the control of the tissue-specific expression of the human IB1 gene.

Fructose-rich diet-induced abdominal adipose tissue endocrine dysfunction in normal male rats.

We have currently studied the changes induced by administration of a fructose-rich diet (FRD) to normal rats in the mass and the endocrine function of abdominal (omental) adipose tissue (AAT). Rats were fed ad libitum a standard commercial chow and tap water, either alone (control diet, CD) or containing fructose (10%, w/vol) (FRD). Three weeks after treatment, circulating metabolic markers and leptin release from adipocytes of AAT were measured. Plasma free fatty acids (FFAs), leptin, adiponectin, and plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in FRD than in CD rats. AAT mass was greater in FRD than in CD rats and their adipocytes were larger, they secreted more leptin and showed impaired insulin sensitivity. While leptin mRNA expression increased in AAT from FRD rats, gene expression of insulin receptor substrate, IRS1 and IRS2 was significantly reduced. Our study demonstrates that administration of a FRD significantly affects insulin sensitivity and several AAT endocrine/metabolic functions. These alterations could be part of a network of interacting abnormalities triggered by FRD-induced oxidative stress at the AAT level. In view of the impaired glucose tolerance observed in FRD rats, these alterations could play a key role in both the development of metabolic syndrome (MS) and beta-cell failure.

Functional analysis of different haematopoietic stem cell subsets

RÉSUMÉ : Elucider les bases moléculaires et cellulaires du fonctionnement des cellules souches s'avère crucial dans la compréhension de l'organisation cellulaire au sein des tissus et des organes ainsi que pour le développement de nouvelles stratégies thérapeutiques en médecine régénérative et en oncologie. Les cellules souches adultes les mieux connues sont celles responsables de l'hématopoïèse, les cellules souches hématopoïétiques (CSH). Durant ces dernières années, la recherche a porté une attention particulière à l'isolation prospective de CSH dérivées de la moelle osseuse de souris en utilisant des marqueurs de surface cellulaire ainsi que des propriétés fonctionnelles alléguées. Par la suite, la capacité fonctionnelle des CSH a été vérifiée classiquement par leur transplantation intraveineuse dans des souris réceptrices conditionnées et par l'analyse de leur aptitude à reconstituer le système hématopoïétique à long terme. Des études récentes suggèrent que la transplantation des cellules directement dans la moelle osseuse pourrait non seulement aboutir à une prise de greffe plus rapide et plus efficace, mais pourrait même aider à l'identification de cellules qui ont certes des propriétés intrinsèques de CSH, mais qui n'ont pas la capacité de trouver leur niche au sein de la moelle osseuse et ont donc échoué dans les analyses classiques de reconstitution. Dans cette étude, nous comparons à deux niveaux la fonction de différents sous-groupes de cellules souches de la moelle osseuse, définis par leur phénotype de surface cellulaire. Premièrement, nous étudions leur capacité à reconstituer des souris létalement irradiées après injection soit intraveineuse soit intrafémorale. Deuxièmement, par analyse cytométrique de flux à 8 couleurs, nous comparons leur activité relative de « side population » (SP) par exclusion du colorant fluorescent Hoechst 33342. Nos résultats préliminaires renforcent en effet l'idée que la transplantation intrafémorale aboutit à une greffe plus rapide et plus efficace. Par contre, en utilisant cette approche, nous n'arrivons pas à identifier des cellules capables de prendre greffe spécifiquement quand elles sont injectées en intrafémorale. Finalement, bien qu'une confirmation in vivo soit encore nécessaire, nous suggérons sur la base de nos analyses cytométriques de flux, que les cellules SP Sca1t~és éie~~ CD48t~és bas sont très enrichies en CSH. Ceci permettrait l'isolation ex vivo de CSH de la moelle osseuse de souris par une stratégie à la fois nouvelle et simple. SUMMARY : Elucidating the molecular and cellular bases of stem cell function is crucial for the understanding of cellular organisation within tissues and organs as well as for the development of new therapeutic strategies in regenerative medicine and oncology. The best-known adult stem cells are those responsible for haematopoiesis, the haematopoietic stem cells (HSCs). In recent years, much effort has been put into the prospective isolation of mouse bone marrow (BM)-derived HSCs using cell-surface markers and alleged functional properties. Upon isolation, the functional capacity of putative HSCs has been classically assessed by intravenous transplantation into conditioned recipient mice and analysis of their ability to reconstitute the haematopoietic system at long-term. It has recently been suggested that transplanting the cells directly into the BM might not only result in more rapid and more effective engraftment, but even help to identify cells that have intrinsic HSC properties but lack the ability to home to their BM niche and have thus failed to succeed in classical reconstitution assays. In this study, we compare the function of different BM cell subsets, as defined by their cell surface phenotype, on two levels. Firstly, we assess their ability to reconstitute lethally irradiated mice, when injected either intravenously or intrafemorally. Secondly, using 8-colour flow cytometric analysis, we compare their relative side population (SP) activity by exclusion of the fluorescent dye Hoechst 33342. Our preliminary results indeed reinforce the idea that intrafemoral transplantation results in faster and more effective engraftment, however, using this approach, we are unable to identify cells that are capable of engrafting specifically when injected intrafemorally. Finally, although in vivo confirmation is still required, we propose, based on the results of our flow cytometric analyses, that SP Scat Very h'9h CD48Very'°W cells should be highly enriched for HSCs. This would allow for a simple new strategy for the isolation of mouse BM HSCs ex vivo.