Role of the REST/RE-1 system in beta-cell life, function and differentiation

SUMMARYDiabetes is characterized by insulin deficiency that results from the destruction of insulin-secreting pancreatic beta-cells (Type 1), or in part from beta-cell death and insulin secretion defects (Type 2). Therefore, understanding the mechanisms of beta cell neogenesis (to generate unlimited supply of beta cells for T1D transplantation] or identifying the specific genes that favors insulin secretion or beta-cell survival is of great importance for the management of diabetes. The transcriptional repressor RE-1 Silencing Transcription Factor (REST) restricts the expression of a large number of genes containing its binding element, called Repressor Element-1 (RE-1), to neurons and beta cells. To do so, REST is ubiquitously expressed but in neurons and beta cells. To identify these essential genes and their functional significance in beta cells, we have generated transgenic mice that express REST specifically in beta cells under the control of the rat insulin promoter (RIP-REST mice). This resulted in the repression of the RE-1- containing genes in beta cells, and we analyzed the consequences.We first showed that RIP-REST mice were glucose-intolerant because of a defective insulin secretion. To explain this defect, we identified that a subset of the REST target genes were necessary for insulin exocytosis, such as Snap25, Synaptotagmin (Syt) IX, Complexin II, and Ica512, and we further demonstrated that among the identified REST targets, Syt IV and VII were also involved in insulin release. We next analyzed a novel RIP-REST mouse line that featured diabetes and we showed that this defect was due to a major loss of beta-cell mass. To explain this phenotype, we identified REST target genes that were involved in beta-cell survival, such as Ibl, Irs2, Ica512 and Connexin36, and revealed that another REST target, Cdk5r2 is also involved in beta-cell protection. In a third part, we finally suggest that REST may be important for pancreatic endocrine differentiation, since transgenic mice expressing constitutive REST in pancreatic multipotent progenitors show impaired formation of Ngn3-expressing endocrine- committed precursors, and impaired formation of differentiated endocrine cells. Mapping the pattern of REST expression in wild type animals indicates that it is expressed in multipotent progenitors to become then excluded from endocrine cells. Preliminary results suggest that a downregulation of REST would result in relieved expression of at least the Mytl target, favoring subsequent acquisition of the endocrine competence by endocrine precursor cells.Thus, we propose that the REST/RE-1 system is an important feature for beta-cell neogenesis, function and survivalRESUMELe diabète se caractérise par une déficience en insuline qui résulte d'une destruction des cellules bêta (β) pancréatiques sécrétant l'insuline [Type 1], ou à un défaut de sécrétion d'insuline qui peut être associé à la mort des cellules β (Type 2). La compréhension des mécanismes de néogenèse des cellules β, ainsi que l'identification de gènes impliqués dans leur survie et dans le contrôle de la sécrétion d'insuline est donc importante pour le traitement du diabète. Le facteur de transcription de type répresseur, RE-1 Silencing Transcription Factor [REST], contribue à la spécificité d'expression dans les neurones et les cellules β, d'un grand nombre de gènes portant son motif de fixation, le Repressor Element-1 (RE-1). Pour cela, REST est exprimé dans toutes les cellules, sauf dans les neurones et les cellules β. Afin d'identifier les gènes cibles de REST ainsi que leur fonction au sein de la cellule β, nous avons généré des souris transgéniques qui expriment REST spécifiquement dans ces cellules, sous la dépendance du promoteur de l'insuline (souris RIP-REST]. Cette expression ectopique de REST a permis de diminuer l'expression des gènes contrôlés par REST, et d'en analyser les conséquences. Nous avons montré que les souris RIP-REST étaient intolérantes au glucose et que ceci était du à un défaut de sécrétion d'insuline. Pour expliquer ce phénotype, nous avons mis en évidence le fait que des gènes cibles de REST codent pour des protéines importantes pour l'exocytose de l'insuline, comme SNAP25, Synaptotagmin (Syt) IX, Complexin II ou ICA512. De plus, nous avons découvert deux nouvelles cibles de REST impliquées dans la sécrétion d'insuline, Syt IV et Syt VII. Par la suite, nous avons démontré qu'une nouvelle lignée de souris RIP-REST étaient atteintes d'un diabète sévère à cause d'une perte massive des cellules β. La disparition de ces cellules a été expliquée par l'identification de gènes cibles de REST impliqués dans la survie des cellules β, comme Ibl, Irs2, Ica512 ou la Connexine36. De plus, nous avons découvert qu'une nouvelle cible, Cdk5r2, était aussi impliquée dans la survie des cellules β. Dans une dernière partie, nous suggérons, grâce à l'analyse de nouvelles souris transgéniques exprimant constitutivement REST dans les cellules progénitrices du pancréas embryonnaire, que REST empêche la formation des précurseurs de cellules endocrines ainsi que la différenciation de ces cellules. L'analyse de l'expression de REST au cours du développement embryonnaire du pancréas indique que la diminution de l'expression de REST conduit en partie, à l'induction d'un de ses gènes cible Mytl, qui favorise la formation de précurseurs endocrines. Nous proposons donc que le système REST/RE-1 est important pour la génération, la fonction et la survie des cellules β.

Selective requirement for c-Myc at an early stage of V(alpha)14i NKT cell development.

Valpha14 invariant (Valpha14i) NKT cells are a subset of regulatory T cells that utilize a semi-invariant TCR to recognize glycolipids associated with monomorphic CD1d molecules. During development in the thymus, CD4(+)CD8(+) Valpha14i NKT precursors recognizing endogenous CD1d-associated glycolipids on other CD4(+)CD8(+) thymocytes are selected to undergo a maturation program involving sequential expression of CD44 and NK-related markers such as NK1.1. The molecular requirements for Valpha14i NKT cell maturation, particularly at early developmental stages, remain poorly understood. In this study, we show that CD4-Cre-mediated T cell-specific inactivation of c-Myc, a broadly expressed transcription factor with a wide range of biological activities, selectively impairs Valpha14i NKT cell development without perturbing the development of conventional T cells. In the absence of c-Myc, Valpha14i NKT cell precursors are blocked at an immature CD44(low)NK1.1(-) stage in a cell autonomous fashion. Residual c-Myc-deficient immature Valpha14i NKT cells appear to proliferate normally, cannot be rescued by transgenic expression of BCL-2, and exhibit characteristic features of immature Valpha14i NKT cells such as high levels of preformed IL-4 mRNA and the transcription factor promyelocytic leukemia zinc finger. Collectively our data identify c-Myc as a critical transcription factor that selectively acts early in Valpha14i NKT cell development to promote progression beyond the CD44(low)NK1.1(-) precursor stage.

Ligand-dependent inhibition of CD1d-restricted NKT cell development in mice transgenic for the activating receptor Ly49D.

In addition to their CD1d-restricted T cell receptor (TCR), natural killer T (NKT) cells express various receptors normally associated with NK cells thought to act, in part, as modulators of TCR signaling. Immunoreceptor-tyrosine activation (ITAM) and inhibition (ITIM) motifs associated with NK receptors may augment or attenuate perceived TCR signals respectively, potentially influencing NKT cell development and function. ITIM-containing Ly49 family receptors expressed by NKT cells are proposed to play a role in their development and function. We have produced mice transgenic for the ITAM-associated Ly49D and ITIM-containing Ly49A receptors and their common ligand H2-Dd to determine the importance of these signaling interplays in NKT cell development. Ly49D/H2-Dd transgenic mice had selectively and severely reduced numbers of thymic and peripheral NKT cells, whereas both ligand and Ly49D transgenics had normal numbers of NKT cells. CD1d tetramer staining revealed a blockade of NKT cell development at an early precursor stage. Coexpression of a Ly49A transgene partially rescued NKT cell development in Ly49D/H2-Dd transgenics, presumably due to attenuation of ITAM signaling. Thus, Ly49D-induced ITAM signaling is incompatible with the early development of cells expressing semi-invariant CD1d-restricted TCRs and appropriately harmonized ITIM-ITAM signaling is likely to play an important role in the developmental program of NKT cells.

Positive impact of inhibitory Ly49 receptor-MHC class I interaction on NK cell development.

NK cells can kill MHC-different or MHC-deficient but not syngeneic MHC-expressing target cells. This MHC class I-specific tolerance is acquired during NK cell development. MHC recognition by murine NK cells largely depends on clonally distributed Ly49 family receptors, which inhibit NK cell function upon ligand engagement. We investigated whether these receptors play a role for the development of NK cells and provide evidence that the expression of a Ly49 receptor transgene on developing NK cells endowed these cells with a significant developmental advantage over NK cells lacking such a receptor, but only if the relevant MHC ligand was present in the environment. The data suggest that the transgenic Ly49 receptor accelerates and/or rescues the development of NK cells which would otherwise fail to acquire sufficient numbers of self-MHC-specific receptors. Interestingly, the positive effect on NK cell development is most prominent when the MHC ligand is simultaneously present on both hemopoietic and nonhemopoietic cells. These findings correlate with functional data showing that MHC class I ligand on all cells is required to generate functionally mature NK cells capable of reacting to cells lacking the respective MHC ligand. We conclude that the engagement of inhibitory MHC receptors during NK cell development provides signals that are important for further NK cell differentiation and/or maturation.

Calmodulin-dependent protein kinase IV during T-cell development.

In a primary cell culture system of fetal rat brain, the calmodulin-dependent protein-kinase IV (CaMKIV) could be induced by the thyroid hormone T3 in a time- and concentration-dependent manner, provided the tissue was excised not later than day 15 of gestation (E15) (Krebs et al., J. Biol. Chem. 271, 11055, 1996). We report here that in the fetal thymus CaMKIV could not be detected earlier than day 16 of gestation and that the expression of this enzyme was fully upregulated at day 18. In mouse fetal thymus organ culture (FTOC) of day 14 embryonic thymus, CaMKIV could not be detected, even after several days of culture if a minimal culture medium lacking fetal calf serum was used. However, after addition of fetal calf serum to the culture medium the expression of CaMKIV could be specifically induced. Furthermore, it could also be shown that during T-cell development in the adult murine thymus the expression of CaMKIV was tightly regulated. Taken together, these results demonstrate that the expression of CaMKIV, an enzyme involved in the regulation of Ca(2+)-dependent gene expression, is itself under stringent regulatory control during tissue development.

Jagged1-dependent Notch signaling is dispensable for hematopoietic stem cell self-renewal and differentiation.

Jagged1-mediated Notch signaling has been suggested to be critically involved in hematopoietic stem cell (HSC) self-renewal. Unexpectedly, we report here that inducible Cre-loxP-mediated inactivation of the Jagged1 gene in bone marrow progenitors and/or bone marrow (BM) stromal cells does not impair HSC self-renewal or differentiation in all blood lineages. Mice with simultaneous inactivation of Jagged1 and Notch1 in the BM compartment survived normally following a 5FU-based in vivo challenge. In addition, Notch1-deficient HSCs were able to reconstitute mice with inactivated Jagged1 in the BM stroma even under competitive conditions. In contrast to earlier reports, these data exclude an essential role for Jagged1-mediated Notch signaling during hematopoiesis.

Rapid IL-4 production by Leishmania homolog of mammalian RACK1-reactive CD4(+) T cells in resistant mice treated once with anti-IL-12 or -IFN-gamma antibodies at the onset of infection with Leishmania major instructs Th2 cell development, resulting in nonhealing lesions.

Rapid production of IL-4 by Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4(+) T cells expressing the V beta 4-V alpha 8 TCR chains has been shown to drive aberrant Th2 cell development and susceptibility to Leishmania major in BALB/c mice. In contrast, mice from resistant strains fail to express this early IL-4 response. However, administration of either anti-IL-12 or -IFN-gamma at the initiation of infection allows the expression of this early IL-4 response in resistant mice. In this work we show that Leishmania homolog of mammalian RACK1-reactive CD4(+) T cells also expressing the V beta 4-V alpha 8 TCR chains are the source of the early IL-4 response to L. major in resistant mice given anti-IL-12 or -IFN-gamma Abs only at the onset of infection. Strikingly, these cells were found to be required for the reversal of the natural resistance of C57BL/6 mice following a single administration of anti-IL-12 or -IFN-gamma Abs. Together these results suggest that a deficiency in mechanisms capable of down-regulating the early IL-4 response to L. major contributes to the exquisite susceptibility of BALB/c mice to L. major.

Targeting PI3KC2β Impairs Proliferation and Survival in Acute Leukemia, Brain Tumours and Neuroendocrine Tumours.

BACKGROUND: Eight human catalytic phosphoinositide 3-kinase (PI3K) isoforms exist which are subdivided into three classes. While class I isoforms have been well-studied in cancer, little is known about the functions of class II PI3Ks. MATERIALS AND METHODS: The expression pattern and functions of the class II PI3KC2β isoform were investigated in a panel of tumour samples and cell lines. RESULTS: Overexpression of PI3KC2β was found in subsets of tumours and cell lines from acute myeloid leukemia (AML), glioblastoma multiforme (GBM), medulloblastoma (MB), neuroblastoma (NB), and small cell lung cancer (SCLC). Specific pharmacological inhibitors of PI3KC2β or RNA interference impaired proliferation of a panel of human cancer cell lines and primary cultures. Inhibition of PI3KC2β also induced apoptosis and sensitised the cancer cells to chemotherapeutic agents. CONCLUSION: Together, these data show that PI3KC2β contributes to proliferation and survival in AML, brain tumours and neuroendocrine tumours, and may represent a novel target in these malignancies.

Cell suspension culture and plant regeneration of a Brazilian plantain, cultivar Terra

The objective of this study was to establish cell suspension culture and plant regeneration via somatic embryogenesis of a Brazilian plantain, cultivar Terra Maranhão, AAB. Immature male flowers were used as explant source for generating highly embryogenic cultures 45 days after inoculation, which were used for establishment of cell suspension culture and multiplication of secondary somatic embryos. Five semisolid culture media were tested for differentiation, maturation, somatic embryos germination and for plant regeneration. An average of 558 plants per one milliliter of 5% SCV (settled cell volume) were regenerated in the MS medium, with 11.4 µM indolacetic acid and 2.2 µM 6-benzylaminopurine. Regenerated plants showed a normal development, and no visible somaclonal variation was observed in vitro. It is possible to regenerate plants from cell suspensions of plantain banana cultivar Terra using MS medium supplemented with 11.4 µM of IAA and 2.2 µM of BAP.

The CD4-like molecule LAG-3, biology and therapeutic applications.

IMPORTANCE OF THE FIELD: Promising immunotherapeutic agents targeting co-stimulatory pathways are currently being tested in clinical trials. One player in this array of regulatory pathways is the LAG-3/MHC class II axis. The lymphocyte activation gene-3 (LAG-3) is a negative co-stimulatory receptor that modulates T cell homeostasis, proliferation and activation. A recombinant soluble dimeric form of LAG-3 (sLAG-3-Ig, IMP321) shows adjuvant properties and enhances immunogenicity of tumor vaccines. Recent clinical trials produced encouraging results, especially when the human dimeric soluble form of LAG-3 (hLAG-3-Ig) was used in combination with chemotherapy. AREAS COVERED IN THIS REVIEW: The biological relevance of LAG-3 in vivo. Pre-clinical data demonstrating adjuvant properties, as well as the improvement of tumor immunity by sLAG-3-Ig. Recent advances in the clinical development of the therapeutic reagent IMP321, hLAG-3-Ig, for cancer treatment. WHAT THE READER WILL GAIN: This review summarizes preclinical and clinical data on the biological functions of LAG-3. TAKE HOME MESSAGE: The LAG-3 inhibitory pathway is attracting attention, in the light of recent studies demonstrating its role in T cell unresponsiveness, and Treg function after chronic antigen stimulation. As a soluble recombinant dimer, the sLAG-3-Ig protein acts as an adjuvant for therapeutic induction of T cell responses, and may be beneficial to cancer patients when used in combination therapies.

Role of the transcription factor sox4 in schwann cell development

Previous studies demonstrated that both Schwann cell differentiation and de-differentiation (in the situation of a nerve injury or demyelinating disease) are regulated by cell-intrinsic regulators including several transcription factors. In particular, the de-differentiation of mature Schwann cells is driven by the activation of multiple negative regulators of myelination including c-Jun, Notch, Sox-2 and Pax-3, all usually expressed in the immature Schwann cells and suppressed at the onset of myelination. In order to identify new negative regulators of myelination involved in the development of the peripheral nervous system (PNS) we analyzed the data from a previously performed transcriptional analysis of myelinating Schwann cells. Based on its transcriptional expression profile during myelination, Sox4, a member of the Sox gene family, was identified as a potential candidate. Previous studies demonstrated that prolonged Sox4 expression in oligodendrocytes maintains these cells in a premyelinating state, further suggesting its role as a negative regulator of myelination. Concomitantly, we observed upregulation of Sox4 mRNA and protein expression levels in the PNS of three different models of demyelinating neuropathies (Pmp22, Lpin1, and Scap KOs). To better characterize the molecular function of Sox4, we used a viral vector allowing Sox4 overexpression in cultured Schwann cells and in neuron-Schwann cell co-cultures. In parallel, we generated two transgenic lines of mice in which the overexpression of Sox4 is driven specifically in Schwann cells by the Myelin Protein Zero gene promoter. The preliminary data from these in vitro and in vivo experiments show that overexpression of Sox4 in PNS causes a delay in progression of myelination thus indicating that Sox4 acts as a negative regulator of Schwann cell myelination.

c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation.

The activity of adult stem cells is essential to replenish mature cells constantly lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. Here, we provide genetic evidence for an unexpected function of the c-Myc protein in the homeostasis of hematopoietic stem cells (HSCs). Conditional elimination of c-Myc activity in the bone marrow (BM) results in severe cytopenia and accumulation of HSCs in situ. Mutant HSCs self-renew and accumulate due to their failure to initiate normal stem cell differentiation. Impaired differentiation of c-Myc-deficient HSCs is linked to their localization in the differentiation preventative BM niche environment, and correlates with up-regulation of N-cadherin and a number of adhesion receptors, suggesting that release of HSCs from the stem cell niche requires c-Myc activity. Accordingly, enforced c-Myc expression in HSCs represses N-cadherin and integrins leading to loss of self-renewal activity at the expense of differentiation. Endogenous c-Myc is differentially expressed and induced upon differentiation of long-term HSCs. Collectively, our data indicate that c-Myc controls the balance between stem cell self-renewal and differentiation, presumably by regulating the interaction between HSCs and their niche.

BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population.

The tumor necrosis factor (TNF) family member B cell activating factor (BAFF) binds B cells and enhances B cell receptor-triggered proliferation. We find that B cell maturation antigen (BCMA), a predicted member of the TNF receptor family expressed primarily in mature B cells, is a receptor for BAFF. Although BCMA was previously localized to the Golgi apparatus, BCMA was found to be expressed on the surface of transfected cells and tonsillar B cells. A soluble form of BCMA, which inhibited the binding of BAFF to a B cell line, induced a dramatic decrease in the number of peripheral B cells when administered in vivo. Moreover, culturing splenic cells in the presence of BAFF increased survival of a percentage of the B cells. These results are consistent with a role for BAFF in maintaining homeostasis of the B cell population.

Homeostatic proliferation and survival of naïve and memory T cells.

The immune system relies on homeostatic mechanisms in order to adapt to the changing requirements encountered during steady-state existence and activation by antigen. For T cells, this involves maintenance of a diverse repertoire of naïve cells, rapid elimination of effector cells after pathogen clearance, and long-term survival of memory cells. The reduction of T-cell counts by either cytotoxic drugs, irradiation, or certain viruses is known to lead to lymphopenia-induced proliferation and restoration of normal T-cell levels. Such expansion is governed by the interaction of TCR with self-peptide/MHC (p/MHC) molecules plus contact with cytokines, especially IL-7. These same ligands, i.e. p/MHC molecules and IL-7, maintain naïve T lymphocytes as resting cells under steady-state T-cell-sufficient conditions. Unlike naïve cells, typical "central" memory T cells rely on a combination of IL-7 and IL-15 for their survival in interphase and for occasional cell division without requiring signals from p/MHC molecules. Other memory T-cell subsets are less quiescent and include naturally occurring activated memory-phenotype cells, memory cells generated during chronic viral infections, and effector memory cells. These subsets of activated memory cells differ from central memory T cells in their requirements for homeostatic proliferation and survival. Thus, the factors controlling T-cell homeostasis can be seen to vary considerably from one subset to another as described in detail in this review.

Cell Cycle Proteins and Retinal Degeneration: Evidences of New Potential Therapeutic Targets.

During different forms of neurodegenerative diseases, including the retinal degeneration, several cell cycle proteins are expressed in the dying neurons from Drosophila to human revealing that these proteins are a hallmark of neuronal degeneration. This is true for animal models of Alzheimer's, and Parkinson's diseases, Amyotrophic Lateral Sclerosis and for Retinitis Pigmentosa as well as for acute injuries such as stroke and light damage. Longitudinal investigation and loss-of-function studies attest that cell cycle proteins participate to the process of cell death although with different impacts, depending on the disease. In the retina, inhibition of cell cycle protein action can result to massive protection. Nonetheless, the dissection of the molecular mechanisms of neuronal cell death is necessary to develop adapted therapeutic tools to efficiently protect photoreceptors as well as other neuron types.