Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon

Emmanouilidou, Evaggelia; Teschemacher, Anja G.; Pouli, Aristea E.; Nicholls, Linda I.; Seward, Elizabeth P.; Rutter, Guy A.

doi:10.1016/s0960-9822(99)80398-4

Cited by 83 publications

(53 citation statements)

References 17 publications

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“…Expression vectors directing the synthesis of miR-124a2 were prepared either by introducing oligonucleotides corresponding to the murine precursor sequence of mir-124a2 into the pcDNA6.2 (Invitrogen), or into human growth hormone expressing vector (pXGH5) (29). In the former case, the oligonucleotide sequences were as follows: sense, 5Ј-TGCTGATC-AAGATCAGAGACTCTGCTCTCCGTGTTCACAGCGGA-CCTTGATTTAATGTCATACAATTAAGGCACGCGGTG-AATGCCAAGAGCGGAGCCTACGGCTGCACTTGAA-3Ј and antisense, 5Ј-CCTGTTCAAGTGCAGCCGTAGGCTCC-GCTCTTGGCATTCACCGCGTGCCTTAATTGTATGAC-ATTAAATCAAGGTCCGCTGTGAACACGGAGAGCGA-GATCTCTGATCTTGATC-3Ј.…”

Section: Methodsmentioning

confidence: 99%

MicroRNA-124a Regulates Foxa2 Expression and Intracellular Signaling in Pancreatic β-Cell Lines

Baroukh

Ravier

Loder

et al. 2007

Journal of Biological Chemistry

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323

309

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MicroRNAs (miRNAs) are short non-coding RNAs that have been implicated in fine-tuning gene regulation, although the precise roles of many are still unknown. Pancreatic development is characterized by the complex sequential expression of a gamut of transcription factors. We have performed miRNA expression profiling at two key stages of mouse embryonic pancreas development, e14.5 and e18.5. miR-124a2 expression was strikingly increased at e18.5 compared with e14.5, suggesting a possible role in differentiated ␤-cells. Among the potential miR124a gene targets identified by biocomputation, Foxa2 is known to play a role in ␤-cell differentiation. To evaluate the impact of miR-124a2 on gene expression, we overexpressed or down-regulated miR-124a2 in MIN6 ␤-cells. As predicted, miR-124a2 regulated Foxa2 gene expression, and that of its downstream target, pancreatic duodenum homeobox-1 (Pdx-1). Foxa2 has been described as a master regulator of pancreatic development and also of genes involved in glucose metabolism and insulin secretion, including the ATP-sensitive K ؉ (K ATP ) channel subunits, Kir6.2 and Sur-1. Correspondingly, miR-124a2 overexpression decreased, and anti-miR-124a2 increased Kir6.2 and Sur-1 mRNA levels. Moreover, miR-124a2 modified basal and glucose-or KCl-stimulated intracellular free Ca 2؉ concentrations in single MIN6 and INS-1 (832/13) ␤-cells, without affecting the secretion of insulin or co-transfected human growth hormone, consistent with an altered sensitivity of the ␤-cell exocytotic machinery to Ca 2؉ . In conclusion, whereas the precise role of microRNA-124a2 in pancreatic development remains to be deciphered, we identify it as a regulator of a key transcriptional protein network in ␤-cells responsible for modulating intracellular signaling.Studies implicating small regulatory RNAs in the control of gene expression have demonstrated that transcriptional regulation is controlled not only by protein factors, but also by small endogenous RNA molecules of ϳ19 -23 nucleotides in length called microRNAs (miRNAs or miRs) 3 (1, 2). The first miRNAs discovered were lin-4 and let-7, which are crucial for regulating the developmental timing in the nematode, Caenorhabditis elegans (1, 3). Since this initial report, several hundred miRNAs have been identified in plants and animals that regulate diverse biological processes ranging from cell metabolism to cell differentiation and growth, apoptosis, and immune responses (4 -8). Moreover, it has been shown that miRNAs are characterized by differential spatial and temporal expression patterns supporting their role in such processes (3, 9). miRNAs serve as regulators of gene expression by binding to complementary sites on their target transcripts and, by an ill-defined mechanism, significantly induce the cleavage of mRNA or the repression of translation, depending on the partial or complete sequence homology, respectively (2, 10 -12). It has been estimated that miRNA genes represent ϳ1% of the genome of complex organisms. It appears that they share a certain...

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Section: Methodsmentioning

confidence: 99%

MicroRNA-124a Regulates Foxa2 Expression and Intracellular Signaling in Pancreatic β-Cell Lines

Baroukh

Ravier

Loder

et al. 2007

Journal of Biological Chemistry

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323

309

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“…2) or depletion (Fig. 4) of insulin gene expression, the release of cotransfected human growth hormone, used as surrogate for insulin (36), was unaffected by the expression of either wild-type or kinase-inactive PASK (Fig. 5A).…”

Section: Changes In Pask Activity Have No Effect On Glucose-stimulatementioning

confidence: 99%

Involvement of Per–Arnt–Sim (PAS) kinase in the stimulation of preproinsulin and pancreatic duodenum homeobox 1 gene expression by glucose

Xavier

Rutter

2004

Proc. Natl. Acad. Sci. U.S.A.

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Per-Arnt-Sim (PAS) domain-containing kinases are common in prokaryotes, but a mammalian counterpart has only recently been described. Although the PAS domain of the mammalian PAS kinase (PASK) is closely related to the bacterial oxygen sensor FixL, it is unclear whether PASK activity is changed in mammalian cells in response to nutrients and might therefore contribute to signal transduction by these or other stimuli. Here, we show that elevated glucose concentrations rapidly increase PASK activity in pancreatic islet ␤ cells, an event followed by the accumulation of both PASK mRNA and protein. Demonstrating a physiological role for PASK activation, comicroinjection into clonal ␤ cells of cDNA encoding wild-type PASK, or PASK protein itself, mimics the induction of preproinsulin promoter activity by high glucose concentrations. Conversely, anti-PASK antibodies block promoter activation by the sugar, and the silencing of PASK expression by RNA interference suppresses the up-regulation by glucose of preproinsulin and pancreatic duodenum homeobox 1 gene expression, without affecting glucose-induced changes in the levels of mRNAs encoding glucokinase or uncoupling protein 2. We conclude that PASK is an important metabolic sensor in nutrient-sensitive mammalian cells and plays an unexpected role in the regulation of key genes involved in maintaining the differentiated phenotype of pancreatic ␤ cells.

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“…The plasmid Gsn-RNAi, previously shown by our group to successfully knock down gelsolin in MIN6 B1 cells (6), was used along with the control plasmid Control-RNAi, encoding for a nonspecific shRNA of nonmammalian origin. Because the transfection efficiency with these plasmids is poor (5-10%) in MIN6 cells, transfected cells were tagged by co-transfection with a plasmid coding phogrin-GFP fusion protein, thereby targeting GFP to the secretory granules and preventing any nuclear localization (26). Preliminary experiments indicated that expression of GFP itself, rather than the fusion protein, interfered with the TUNEL assay because of the presence of GFP in the nucleus.…”

Section: Induction Of Apoptosis In Min6 B1 and C3 Cellsmentioning

confidence: 99%

Pro-Survival Role of Gelsolin in Mouse β-Cells

Yermen¹,

Tomás²,

Halban³

2007

Diabetes

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We have previously shown that the Ca 2؉ -dependent actinsevering protein gelsolin plays an important role in regulated insulin secretion. The aim of this study was to determine the role of gelsolin in ␤-cell survival as it has been shown to play a dual role in apoptosis in other cell types. MIN6 subclones B1 and C3, shown previously to express gelsolin at different levels (B1Ͼ ϾC3 cells), were used for this purpose. We demonstrate that B1 cells have lower levels of apoptosis and active caspase-3 when compared with C3 cells, in both standard (25 mmol/l glucose and 15% FCS) and deprived (5 mmol/l glucose and 1% FCS) conditions. Overexpression of gelsolin resulted in a decrease in the percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)

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Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon

Cited by 83 publications

References 17 publications

MicroRNA-124a Regulates Foxa2 Expression and Intracellular Signaling in Pancreatic β-Cell Lines

MicroRNA-124a Regulates Foxa2 Expression and Intracellular Signaling in Pancreatic β-Cell Lines

Involvement of Per–Arnt–Sim (PAS) kinase in the stimulation of preproinsulin and pancreatic duodenum homeobox 1 gene expression by glucose

Pro-Survival Role of Gelsolin in Mouse β-Cells

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