Virus-like infection induces human ß cell dedifferentiation.

Type 1 diabetes (T1D) is a chronic disease characterized by an autoimmune-mediated destruction of insulin-producing pancreatic ß cells. Environmental factors such as viruses play an important role in the onset of T1D and interact with predisposing genes. Recent data suggest that viral infection of human islets leads to a decrease in insulin production rather than ß cell death, suggesting loss of ß cell identity. We undertook this study to examine whether viral infection could induce human ß cell dedifferentiation. Using the functional human ß cell line EndoC-ßH1, we demonstrate that polyinosinic-polycytidylic acid (PolyI:C), a synthetic double-stranded RNA that mimics a byproduct of viral replication, induces a decrease in ß cell-specific gene expression. In parallel with this loss, the expression of progenitor-like genes such as SOX9 was activated following PolyI:C treatment or enteroviral infection. SOX9 was induced by the NF-κB pathway and also in a paracrine non-cell-autonomous fashion through the secretion of IFN-α. Lastly, we identified SOX9 targets in human ß cells as potentially new markers of dedifferentiation in T1D. These findings reveal that inflammatory signaling has clear implications in human ß cell dedifferentiation.

Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs.

During somatic hypermutation (SHM) of immunoglobulin genes, uracils introduced by activation-induced cytidine deaminase are processed by uracil-DNA glycosylase (UNG) and mismatch repair (MMR) pathways to generate mutations at G-C and A-T base pairs, respectively. Paradoxically, the MMR-nicking complex Pms2/Mlh1 is apparently dispensable for A-T mutagenesis. Thus, how detection of U:G mismatches is translated into the single-strand nick required for error-prone synthesis is an open question. One model proposed that UNG could cooperate with MMR by excising a second uracil in the vicinity of the U:G mismatch, but it failed to explain the low impact of UNG inactivation on A-T mutagenesis. In this study, we show that uracils generated in the G1 phase in B cells can generate equal proportions of A-T and G-C mutations, which suggests that UNG and MMR can operate within the same time frame during SHM. Furthermore, we show that Ung-/-Pms2-/- mice display a 50% reduction in mutations at A-T base pairs and that most remaining mutations at A-T bases depend on two additional uracil glycosylases, thymine-DNA glycosylase and SMUG1. These results demonstrate that Pms2/Mlh1 and multiple uracil glycosylases act jointly, each one with a distinct strand bias, to enlarge the immunoglobulin gene mutation spectrum from G-C to A-T bases.

Extracellular acidification stimulates GPR68 mediated IL-8 production in human pancreatic ß cells.

Acute or chronic metabolic complications such as diabetic ketoacidosis are often associated with extracellular acidification and pancreatic ß-cell dysfunction. However, the mechanisms by which human ß-cells sense and respond to acidic pH remain elusive. In this study, using the recently developed human ß-cell line EndoC-ßH2, we demonstrate that ß-cells respond to extracellular acidification through GPR68, which is the predominant proton sensing receptor of human ß-cells. Using gain- and loss-of-function studies, we provide evidence that the ß-cell enriched transcription factor RFX6 is a major regulator of GPR68. Further, we show that acidic pH stimulates the production and secretion of the chemokine IL-8 by ß-cells through NF-кB activation. Blocking of GPR68 or NF-кB activity severely attenuated acidification induced IL-8 production. Thus, we provide mechanistic insights into GPR68 mediated ß-cell response to acidic microenvironment, which could be a new target to protect ß-cell against acidosis induced inflammation.

Ex Vivo Expansion and Differentiation of Human and Mouse Fetal Pancreatic Progenitors Are Modulated by Epidermal Growth Factor.

A comparative analysis of mouse and human pancreatic development may reveal common mechanisms that control key steps as organ morphogenesis and cell proliferation and differentiation. More specifically, understanding beta cell development remains an issue, despite recent progress related to their generation from human embryonic and induced pluripotent stem cells. In this study, we use an integrated approach, including prospective isolation, organ culture, and characterization of intermediate stages, and report that cells from human and mouse fetal pancreas can be expanded in the long term and give rise to hollow duct-like structures in 3D cultures. The expanded cells express a combination of markers (E-cadherin, PDX1, NKX6-1, SOX9, and HNF1ß) that reveals pancreatic progenitor identity. Proliferation of embryonic progenitors was stimulated by the Wnt agonist R-spondin1 (RSPO1), FGF10, and EGF. This combination of growth factors allowed maintaining human fetal pancreatic progenitors in culture for many passages, a finding not reported previously. Importantly, in the absence of EGF, proliferation was reduced, while endocrine differentiation was significantly enhanced. We conclude that modulation of EGF signaling affects in vitro expansion and differentiation of progenitors from embryonic pancreas of both mice and man.

RFX6 regulates insulin secretion by modulating Ca2+ homeostasis in human ß cells.

Development and function of pancreatic ß cells involve the regulated activity of specific transcription factors. RFX6 is a transcription factor essential for mouse ß cell differentiation that is mutated in monogenic forms of neonatal diabetes. However, the expression and functional roles of RFX6 in human ß cells, especially in pathophysiological conditions, are poorly explored. We demonstrate the presence of RFX6 in adult human pancreatic endocrine cells. Using the recently developed human ß cell line EndoC-ßH2, we show that RFX6 regulates insulin gene transcription, insulin content, and secretion. Knockdown of RFX6 causes downregulation of Ca(2+)-channel genes resulting in the reduction in L-type Ca(2+)-channel activity that leads to suppression of depolarization-evoked insulin exocytosis. We also describe a previously unreported homozygous missense RFX6 mutation (p.V506G) that is associated with neonatal diabetes, which lacks the capacity to activate the insulin promoter and to increase Ca(2+)-channel expression. Our data therefore provide insights for understanding certain forms of neonatal diabetes.

A new generation of pPRIG-based retroviral vectors.

BACKGROUND: Retroviral vectors are valuable tools for gene transfer. Particularly convenient are IRES-containing retroviral vectors expressing both the protein of interest and a marker protein from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV enhancer-promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient transfection. However, pAP2 contains a limited multicloning site (MCS) and shows weak eGFP expression, which previously led us to engineer an improved version, termed pPRIG, harboring: i) the wild-type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked by T7 and SP6 sequences; and iii) a HA tag encoding sequence 5' of the MCS (pPRIG HAa/b/c). RESULTS: The convenience of pPRIG makes it a good basic vector to generate additional derivatives for an extended range of use. Here we present several novel pPRIG-based vectors (collectively referred to as PRIGs) in which : i) the HA tag sequence was inserted in the three reading frames 3' of the MCS (3'HA PRIGs); ii) a functional domain (ER, VP16 or KRAB) was inserted either 5' or 3' of the MCS (<< modular >> PRIGs); iii) eGFP was replaced by either eCFP, eYFP, mCherry or puro-R (<< single color/resistance >> PRIGs); and iv) mCherry, eYFP or eGFP was inserted 5' of the MCS of the IRES-eGFP, IRES-eCFP or IRES-Puro-R containing PRIGs, respectively (<< dual color/selection >> PRIGs). Additionally, some of these PRIGs were also constructed in a pMigR MSCV background which has been widely used in pluripotent cells. CONCLUSION: These novel vectors allow for straightforward detection of any expressed protein (3'HA PRIGs), for functional studies of chimeric proteins (<< modular >> PRIGs), for multiple transductions and fluorescence analyses of transduced cells (<< single color/resistance >> PRIGs), or for quantitative detection of studied proteins in independently identified/selected transduced cells (<< dual color/selection >> PRIGs). They maintain the original advantages of pPRIG and provide suitable tools for either transient or stable expression and functional studies in a large range of experimental settings.

The relationship between BCL6 bodies and nuclear sites of normal and halogenated DNA and RNA synthesis.

BCL6 is a POZ/BTB and zinc finger transcription factor that self-interacts and accumulates into discrete nuclear "bodies" of unknown function. We recently reported that BCL6 bodies associate with bromodeoxyuridine (BrdU)-substituted DNA, suggesting their implication in replication. To examine this possibility, we examine here by electron and confocal microscopy the relation between BCL6 bodies and replication foci (RF) using incorporation of various halogenated nucleotides (BrdU, chlorodeoxyuridine, CldU, and iododeoxyuridine, IdU) or PCNA (proliferating cell nuclear antigen) staining. We show that BCL6 bodies are found associated with RF, as revealed by PCNA staining. However, such association is markedly prolonged upon BrdU or CldU incorporation, but less, or not at all, upon IdU incorporation. Pulse-chase and double-labeling experiments indicate that IdU-substituted DNA leaves BCL6 bodies after a few tenths of minutes while BrdU- or CldU-substituted DNA stalls in their vicinity for several hours, thereby giving the characteristic "crowns" of DNA entirely surrounding BCL6 bodies. In all cases, however, the halogenated DNA ends up undergoing a movement from BCL6 bodies toward nucleoplasm and nuclear periphery to reach euchromatin and heterochromatin, respectively. We propose that replicating DNA is prone to be bound by BCL6, while BrdU/CldU incorporation increases this propensity possibly because these two events have synergistic effects on the structure and chromatinisation of the newly synthesized DNA. Finally, despite the known proximity between nuclear sites of transcription and replication, we show via several approaches that BCL6 bodies do not appear to be involved either in RNA synthesis or storage.

Ambivalent role of BCL6 in cell survival and transformation.

The BCL6 gene is often structurally altered and probably 'misregulated' in two different types of human B-cell non-Hodgkin lymphomas (BNHL) thought to arise from germinal centre B cells. BCL6 encodes a BTB/POZ and zinc finger protein whose biochemical properties support a role as a DNA-binding transcriptional repressor and disclose, in part, the underlying mechanisms. In contrast, the study of the 'oncogenic' structural alterations of BCL6 in BNHL and of its cellular functions gives rise to much more heterogeneous data with no obvious unifying picture so that how and even whether BCL6 contributes to lymphomagenesis remains unclear. This review will summarize the current knowledge about the 'oncogenic' alterations and cellular functions of BCL6 and, based on some results, will propose the following hypotheses: (1) In various systems, including in memory T cells and also in germinal centre B cells and possibly in certain postmitotic cells, BCL6 may act by stabilizing a particular stage of differentiation. (2) Both its ambivalent effects on cell survival and the heterogeneous consequences of its alterations in BNHL suggest that BCL6 can be oncogenic not only upon overexpression or persistent expression, as often proposed, but also, similar to some of its relatives, upon 'accidental' downregulation.