New α- and SIN γ-retrovectors for safe transduction and specific transgene expression in pancreatic ß cell lines.

BACKGROUND: Viral vectors are invaluable tools to transfer genes and/or regulatory sequences into differentiated cells such as pancreatic cells. To date, several kinds of viral vectors have been used to transduce different pancreatic cell types, including insulin-producing ß cells. However, few studies have used vectors derived from « simple ¼ retroviruses, such as avian α- or mouse γ-retroviruses, despite their high experimental convenience. Moreover, such vectors were never designed to specifically target transgene expression into ß cells. RESULTS: We here describe two novel α- or SIN (Self-Inactivating) γ-retrovectors containing the RIP (Rat Insulin Promoter) as internal promoter. These two retrovectors are easily produced in standard BSL2 conditions, rapidly concentrated if needed, and harbor a large multiple cloning site. For the SIN γ-retrovector, either the VSV-G (pantropic) or the retroviral ecotropic (rodent specific) envelope was used. For the α-retrovector, we used the A type envelope, as its receptor, termed TVA, is only naturally present in avian cells and can efficiently be provided to mammalian ß cells through either exogenous expression upon cDNA transfer or gesicle-mediated delivery of the protein. As expected, the transgenes cloned into the two RIP-containing retrovectors displayed a strong preferential expression in ß over non-ß cells compared to transgenes cloned in their non-RIP (CMV- or LTR-) regulated counterparts. We further show that RIP activity of both retrovectors mirrored fluctuations affecting endogenous INSULIN gene expression in human ß cells. Finally, both α- and SIN γ-retrovectors were extremely poorly mobilized by the BXV1 xenotropic retrovirus, a common invader of human cells grown in immunodeficient mice, and, most notably, of human ß cell lines. CONCLUSION: Our novel α- and SIN γ-retrovectors are safe and convenient tools to stably and specifically express transgene(s) in mammalian ß cells. Moreover, they both reproduce some regulatory patterns affecting INSULIN gene expression. Thus, they provide a helpful tool to both study the genetic control of ß cell function and monitor changes in their differentiation status.

Haploinsufficiency of Dmxl2, encoding a synaptic protein, causes infertility associated with a loss of GnRH neurons in mouse.

Characterization of the genetic defects causing gonadotropic deficiency has made a major contribution to elucidation of the fundamental role of Kisspeptins and Neurokinin B in puberty onset and reproduction. The absence of puberty may also reveal neurodevelopmental disorders caused by molecular defects in various cellular pathways. Investigations of these neurodevelopmental disorders may provide information about the neuronal processes controlling puberty onset and reproductive capacity. We describe here a new syndrome observed in three brothers, which involves gonadotropic axis deficiency, central hypothyroidism, peripheral demyelinating sensorimotor polyneuropathy, mental retardation, and profound hypoglycemia, progressing to nonautoimmune insulin-dependent diabetes mellitus. High-throughput sequencing revealed a homozygous in-frame deletion of 15 nucleotides in DMXL2 in all three affected patients. This homozygous deletion was associated with lower DMXL2 mRNA levels in the blood lymphocytes of the patients. DMXL2 encodes the synaptic protein rabconnectin-3α, which has been identified as a putative scaffold protein for Rab3-GAP and Rab3-GEP, two regulators of the GTPase Rab3a. We found that rabconnectin-3α was expressed in exocytosis vesicles in gonadotropin-releasing hormone (GnRH) axonal extremities in the median eminence of the hypothalamus. It was also specifically expressed in cells expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) within the pituitary. The conditional heterozygous deletion of Dmxl2 from mouse neurons delayed puberty and resulted in very low fertility. This reproductive phenotype was associated with a lower number of GnRH neurons in the hypothalamus of adult mice. Finally, Dmxl2 knockdown in an insulin-secreting cell line showed that rabconnectin-3α controlled the constitutive and glucose-induced secretion of insulin. In conclusion, this study shows that low levels of DMXL2 expression cause a complex neurological phenotype, with abnormal glucose metabolism and gonadotropic axis deficiency due to a loss of GnRH neurons. Our findings identify rabconectin-3α as a key controller of neuronal and endocrine homeostatic processes.

Culture, differentiation, and transduction of mouse E12.5 pancreatic spheres: an in vitro model for the secondary transition of pancreas development.

During the secondary transition of rodent pancreatic development, mainly between E12.5 and E15.5 in mice, exocrine and endocrine populations differentiate from pancreatic progenitors. Here we describe an experimental system for its study in vitro. First, we show that spheres derived from dissociated E12.5 mouse pancreases differentiate within 7 days into most pancreatic exocrine and endocrine cell types, including beta cells. The proportion and spatial repartition of the different endocrine populations mirror those observed during normal development. Thus, dissociation and culture do not impair the developmental events affecting pancreatic progenitors during the secondary transition. Moreover, dissociated cells from mouse E12.5 pancreas were transduced with ecotropic MLV-based retroviral vectors or, though less efficiently, with a mixture of ALV(A)-based retroviral vectors and gesicles containing the TVA (Tumor Virus A) receptor. As an additional improvement, we also created a transgenic mouse line expressing TVA under the control of the 4.5 kB pdx1 promoter (pdx1-TVA). We demonstrate that pancreatic progenitors from dissociated pdx1-TVA pancreas can be specifically transduced by ALV(A)-based retroviral vectors. Using this model, we expressed an activated mutant of the YAP transcriptional co-activator in pancreatic progenitors. These experiments indicate that deregulated YAP activity reduces endocrine and exocrine differentiation in the resulting spheres, confirming and extending previously published data. Thus, our experimental model recapitulates in vitro the crucial developmental decisions arising at the secondary transition and provides a convenient tool to study their genetic control.

Red wine triggers cell death and thioredoxin reductase inhibition: effects beyond resveratrol and SIRT1.

Red wine contains antioxidants and is at moderate amounts believed to exert certain positive health effects. Resveratrol is one of the most studied antioxidants in red wine and has been suggested to activate the longevity- and metabolism-associated histone deacetylase SIRT1. Here we show that relatively low concentrations of resveratrol (0.5-3 microM) specifically inhibited neuronal differentiation of neural stem cells in a SIRT1-dependent manner whereas higher concentrations of resveratrol (> or =10 microM) induced a SIRT1-independent cell death. Surprisingly, using a cell based assay, we found that small amounts of red wine (1-5% v/v)--but not white wine--induced a massive and rapid cell death of various cell types, including neural stem cells and several cancer cell lines. This red wine-induced cell death was ethanol-, SIRT1- and resveratrol-independent but associated with increased oxidative stress and inhibition of thioredoxin reductase (TrxR) activity. The TrxR inhibition correlated with the red color (absorbance at 520 nm) of the wines demonstrating that pigment components of red wine can exert profound cellular effects. Our results unveil important roles for SIRT1 and TrxR in resveratrol and red wine-mediated effects on progenitor and cancer cells, and demonstrate that cellular responses to red wine may be more complex than generally appreciated.

Bromodomain and Extra Terminal Protein Inhibitors Promote Pancreatic Endocrine Cell Fate.

Bromodomain and extraterminal (BET) proteins are epigenetic readers that interact with acetylated lysines of histone tails. Recent studies have demonstrated their role in cancer progression because they recruit key components of the transcriptional machinery to modulate gene expression. However, their role during embryonic development of the pancreas has never been studied. Using mouse embryonic pancreatic explants and human induced pluripotent stem cells (hiPSCs), we show that BET protein inhibition with I-BET151 or JQ1 enhances the number of neurogenin3 (NEUROG3) endocrine progenitors. In mouse explants, BET protein inhibition further led to increased expression of ß-cell markers but in the meantime, strongly downregulated Ins1 expression. Similarly, although acinar markers, such as Cpa1 and CelA, were upregulated, Amy expression was repressed. In hiPSCs, BET inhibitors strongly repressed C-peptide and glucagon during endocrine differentiation. Explants and hiPSCs were then pulsed with BET inhibitors to increase NEUROG3 expression and further chased without inhibitors. Endocrine development was enhanced in explants with higher expression of insulin and maturation markers, such as UCN3 and MAFA. In hiPSCs, the outcome was different because C-peptide expression remained lower than in controls, but ghrelin expression was increased. Altogether, by using two independent models of pancreatic development, we show that BET proteins regulate multiple aspects of pancreatic development.

DNA polymorphisms of the KiSS1 3' untranslated region interfere with the folding of a G-rich sequence into G-quadruplex.

KISS1R and its ligand, the kisspeptins, are key hypothalamic factors that regulate GnRH hypothalamic secretion and therefore the pubertal timing. During studies analysing KiSS1 as a candidate gene in pubertal onset disorders, two SNP and one nucleotide insertion were observed in a 23 nucleotides G-rich sequence located 65 nucleotides downstream of the stop codon. The polymorphisms formed four haplotypes. Biophysical experiments revealed the ability of this G-rich sequence to fold into G-quadruplex structures and demonstrated that the three DNA polymorphisms did not perturb the folding into G-quadruplex but affected G-quadruplex conformation. A functional luciferase reporter-based assay revealed functional differences between 3'UTR haplotypes. These data show that polymorphisms in a G-rich sequence of the 3'UTR of KISS1, able to fold into G-quadruplex structures, can modulate gene expression. They highlight the potential role of this G-quadruplex in the regulation of KISS1 expression and in the timing of pubertal onset.

KISS1 is down-regulated by 17beta-estradiol in MDA-MB-231 cells through a nonclassical mechanism and loss of ribonucleic acid polymerase II binding at the proximal promoter.

Kisspeptins are hypothalamic neuropeptides encoded by KISS1 and recently described as major regulators of GnRH release from hypothalamic neurons. Although 17beta-estradiol (E2)-induced up-regulation of KISS1 expression has been documented in anteroventral periventricular nucleus neurons, E2 down-regulates KISS1 expression in arcuate nucleus neurons via the estrogen receptor alpha by unknown molecular mechanisms. Because KISS1 was initially described as a metastasis inhibitor, notably in breast tumors, we used the MDA-MB-231 breast cancer cell line, which expresses high levels of KISS1, to characterize the molecular mechanism underlying KISS1 regulation by E2. E2 rapidly down-regulated endogenous KISS1 in a stable ERalpha-expressing MDA-MB-231 cell line. Promoter analysis revealed that E2 down-regulation was determined by a short 93-bp sequence devoid of estrogen response element and Sp1 sites. E2 down-regulation persisted with an ERalpha that was unable to bind DNA and in the presence of histone deacetylase inhibitor. In the absence of E2, unliganded ERalpha and RNA polymerase II (RNAPII) were present on the proximal promoter. E2 stimulation induced recruitment of ERalpha and loss of RNAPII at the proximal promoter. Along the gene body, total RNAPII amounts were similar in E2-treated and untreated cells, whereas the active form was significantly less abundant in E2-treated cells. Thus, E2-induced down-regulation of KISS1 is mediated by a pathway combining RNAPII loss at the proximal promoter and modulation of active RNAPII along the gene body, which is a novel mechanism in the complex process of E2-induced repression of gene expression.