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1.  Generation of Insulin-Producing Cells from the Mouse Liver Using β Cell-Related Gene Transfer Including Mafa and Mafb 
PLoS ONE  2014;9(11):e113022.
Recent studies on the large Maf transcription factors have shown that Mafb and Mafa have respective and distinctive roles in β-cell development and maturation. However, whether this difference in roles is due to the timing of the gene expression (roughly, expression of Mafb before birth and of Mafa after birth) or to the specific function of each gene is unclear. Our aim was to examine the functional differences between these genes that are closely related to β cells by using an in vivo model of β-like cell generation. We monitored insulin gene transcription by measuring bioluminescence emitted from the liver of insulin promoter-luciferase transgenic (MIP-Luc-VU) mice. Adenoviral gene transfers of Pdx1/Neurod/Mafa (PDA) and Pdx1/Neurod/Mafb (PDB) combinations generated intense luminescence from the liver that lasted for more than 1 week and peaked at 3 days after transduction. The peak signal intensities of PDA and PDB were comparable. However, PDA but not PDB transfer resulted in significant bioluminescence on day 10, suggesting that Mafa has a more sustainable role in insulin gene activation than does Mafb. Both PDA and PDB transfers ameliorated the glucose levels in a streptozotocin (STZ)-induced diabetic model for up to 21 days and 7 days, respectively. Furthermore, PDA transfer induced several gene expressions necessary for glucose sensing and insulin secretion in the liver on day 9. However, a glucose tolerance test and liver perfusion experiment did not show glucose-stimulated insulin secretion from intrahepatic β-like cells. These results demonstrate that bioluminescence imaging in MIP-Luc-VU mice provides a noninvasive means of detecting β-like cells in the liver. They also show that Mafa has a markedly intense and sustained role in β-like cell production in comparison with Mafb.
PMCID: PMC4232560  PMID: 25397325
2.  MafA Is Required for Postnatal Proliferation of Pancreatic β-Cells 
PLoS ONE  2014;9(8):e104184.
The postnatal proliferation and maturation of insulin-secreting pancreatic β-cells are critical for glucose metabolism and disease development in adults. Elucidation of the molecular mechanisms underlying these events will be beneficial to direct the differentiation of stem cells into functional β-cells. Maturation of β-cells is accompanied by increased expression of MafA, an insulin gene transcription factor. Transcriptome analysis of MafA knockout islets revealed MafA is required for the expression of several molecules critical for β-cell function, including Glut2, ZnT8, Granuphilin, Vdr, Pcsk1 and Urocortin 3, as well as Prolactin receptor (Prlr) and its downstream target Cyclin D2 (Ccnd2). Inhibition of MafA expression in mouse islets or β-cell lines resulted in reduced expression of Prlr and Ccnd2, and MafA transactivated the Prlr promoter. Stimulation of β-cells by prolactin resulted in the phosphorylation and translocation of Stat5B and an increased nuclear pool of Ccnd2 via Prlr and Jak2. Consistent with these results, the loss of MafA resulted in impaired proliferation of β-cells at 4 weeks of age. These results suggest that MafA regulates the postnatal proliferation of β-cells via prolactin signaling.
PMCID: PMC4134197  PMID: 25126749
3.  Generation and Characterization of Ins1-cre-driver C57BL/6N for Exclusive Pancreatic Beta Cell-specific Cre-loxP Recombination 
Experimental Animals  2014;63(2):183-191.
Cre/loxP system-mediated site-specific recombination is utilized to study gene function in vivo. Successful conditional knockout of genes of interest is dependent on the availability of Cre-driver mice. We produced and characterized pancreatic β cell-specific Cre-driver mice for use in diabetes mellitus research. The gene encoding Cre was inserted into the second exon of mouse Ins1 in a bacterial artificial chromosome (BAC). Five founder mice were produced by microinjection of linearized BAC Ins1-cre. The transgene was integrated between Mafa and the telomere on chromosome 15 in one of the founders, BAC Ins1-cre25. To investigate Cre-loxP recombination, BAC Ins1-cre25 males were crossed with two different Cre-reporters, R26R and R26GRR females. On gross observation, reporter signal after Cre-loxP recombination was detected exclusively in the adult pancreatic islets in both F1 mice. Immunohistological analysis indicated that Cre-loxP recombination-mediated reporter signal was colocalized with insulin in pancreatic islet cells of both F1 mice, but not with glucagon. Moreover, Cre-loxP recombination signal was already observed in the pancreatic islets at E13.5 in both F1 fetuses. Finally, we investigated ectopic Cre-loxP recombination for Ins1, because the ortholog Ins2 is also expressed in the brain, in addition to the pancreas. However, there was no Cre-loxP recombination-mediated reporter signal in the brain of both F1 mice. Our data suggest that BAC Ins1-cre25 mice are a useful Cre-driver C57BL/6N for pancreatic β cell-specific Cre-loxP recombination, except for crossing with knock-in mice carrying floxed gene on chromosome 15.
PMCID: PMC4160984  PMID: 24770644
cre-driver mice; cre-loxP recombination; diabetes; insulin1; pancreatic β cells
4.  Bioluminescence Imaging of β Cells and Intrahepatic Insulin Gene Activity under Normal and Pathological Conditions 
PLoS ONE  2013;8(4):e60411.
In diabetes research, bioluminescence imaging (BLI) has been applied in studies of β-cell impairment, development, and islet transplantation. To develop a mouse model that enables noninvasive imaging of β cells, we generated a bacterial artificial chromosome (BAC) transgenic mouse in which a mouse 200-kbp genomic fragment comprising the insulin I gene drives luciferase expression (Ins1-luc BAC transgenic mouse). BLI of mice was performed using the IVIS Spectrum system after intraperitoneal injection of luciferin, and the bioluminescence signal from the pancreatic region analyzed. When compared with MIP-Luc-VU mice [FVB/N-Tg(Ins1-luc)VUPwrs/J] expressing luciferase under the control of the 9.2-kbp mouse insulin I promoter (MIP), the bioluminescence emission from Ins1-luc BAC transgenic mice was enhanced approximately 4-fold. Streptozotocin-treated Ins1-luc BAC transgenic mice developed severe diabetes concomitant with a sharp decline in the BLI signal intensity in the pancreas. Conversely, mice fed a high-fat diet for 8 weeks showed an increase in the signal, reflecting a decrease or increase in the β-cell mass. Although the bioluminescence intensity of the islets correlated well with the number of isolated islets in vitro, the intensity obtained from a living mouse in vivo did not necessarily reflect an absolute quantification of the β-cell mass under pathological conditions. On the other hand, adenovirus-mediated gene transduction of β-cell-related transcription factors in Ins1-luc BAC transgenic mice generated luminescence from the hepatic region for more than 1 week. These results demonstrate that BLI in Ins1-luc BAC transgenic mice provides a noninvasive method of imaging islet β cells and extrapancreatic activity of the insulin gene in the liver under normal and pathological conditions.
PMCID: PMC3617225  PMID: 23593212
5.  Dampened ERK Signaling in Hematopoietic Progenitor Cells in Rheumatoid Arthritis 
In rheumatoid arthritis (RA), hematopoietic progenitor cells (HPC) have age-inappropriate telomeric shortening suggesting premature senescence and possible restriction of proliferative capacity. In response to hematopoietic growth factors RA-derived CD34+ HPC expanded significantly less than age-matched controls. Cell surface receptors for stem cell factor (SCF), Flt 3-Ligand, IL-3 and IL-6 were intact in RA HPC but the cells had lower transcript levels of cell cycle genes, compatible with insufficient signal strength in the ERK pathway. Cytokine-induced phosphorylation of ERK1/2 was diminished in RA HPC whereas phosphorylated STAT3 and STAT5 molecules accumulated to a similar extent as in controls. Confocal microscopy demonstrated that the membrane-proximal colocalization of K-Ras and B-Raf were less efficient in RA-derived CD34+ cells. Thus, hyporesponsiveness of RA HPC to growth factors results from dampening of the ERK signaling pathways; with a defect localized in the very early steps of the ERK signaling cascade.
PMCID: PMC3303971  PMID: 22342385
rheumatoid arthritis; hematopoietic progenitor cells; CD34; ERK signaling; STAT signaling
6.  A Bacterial Artificial Chromosome Transgene with Polymorphic Cd72 Inhibits the Development of Glomerulonephritis and Vasculitis in MRL-Faslpr Lupus Mice 
Systemic lupus erythematosus is considered to be under the control of polygenic inheritance, developing according to the cumulative effects of susceptibility genes with polymorphic alleles; however, the mechanisms underlying the roles of polygenes based on functional and pathological genomics remain uncharacterized. In this study, we substantiate that a CD72 polymorphism in the membrane-distal extracellular domain impacts on both the development of glomerulonephritis and vasculitis in a lupus model strain of mice, MRL/MpJ-Faslpr, and the reactivity of BCR signal stimulation. We generated mice carrying a bacterial artificial chromosome transgene originating from C57BL/6 (B6) mice that contains the Cd72b locus (Cd72B6 transgenic [tg]) or the modified Cd72b locus with an MRL-derived Cd72c allele at the polymorphic region corresponding to the membrane-distal extracellular domain (Cd72B6/MRL tg). Cd72B6 tg mice, but not Cd72B6/MRL tg mice, showed a significant reduction in mortality following a marked improvement of disease associated with decreased serum levels of IgG3 and anti-dsDNA Abs. The number of splenic CD4−CD8− T cells in Cd72B6 tg mice was decreased significantly in association with a reduced response to B cell receptor signaling. These results indicate that the Cd72 polymorphism affects susceptibility to lupus phenotypes and that novel functional rescue by a bacterial artificial chromosome transgenesis is an efficient approach with wide applications for conducting a genomic analysis of polygene diseases.
PMCID: PMC3575566  PMID: 23365086
7.  The E3 Ubiquitin Ligase Activity of Trip12 Is Essential for Mouse Embryogenesis 
PLoS ONE  2011;6(10):e25871.
Protein ubiquitination is a post-translational protein modification that regulates many biological conditions [1], [2], [3], [4]. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1 [5], [6]. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12mt/mt) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12mt/mt embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16 [7], [8], [9], [10]. In contrast, Trip12mt/mt ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12mt/mt ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.
PMCID: PMC3196520  PMID: 22028794
8.  Deficiency of the DNA repair enzyme ATM in rheumatoid arthritis 
The Journal of Experimental Medicine  2009;206(6):1435-1449.
In rheumatoid arthritis (RA), dysfunctional T cells sustain chronic inflammatory immune responses in the synovium. Even unprimed T cells are under excessive replication pressure, suggesting an intrinsic defect in T cell regeneration. In naive CD4 CD45RA+ T cells from RA patients, DNA damage load and apoptosis rates were markedly higher than in controls; repair of radiation-induced DNA breaks was blunted and delayed. DNA damage was highest in newly diagnosed untreated patients. RA T cells failed to produce sufficient transcripts and protein of the DNA repair kinase ataxia telangiectasia (AT) mutated (ATM). NBS1, RAD50, MRE11, and p53 were also repressed. ATM knockdown mimicked the biological effects characteristic for RA T cells. Conversely, ATM overexpression reconstituted DNA repair capabilities, response patterns to genotoxic stress, and production of MRE11 complex components and rescued RA T cells from apoptotic death. In conclusion, ATM deficiency in RA disrupts DNA repair and renders T cells sensitive to apoptosis. Apoptotic attrition of naive T cells imposes lymphopenia-induced proliferation, leading to premature immunosenescence and an autoimmune-biased T cell repertoire. Restoration of DNA repair mechanisms emerges as an important therapeutic target in RA.
PMCID: PMC2715066  PMID: 19451263
9.  MafA Is a Key Regulator of Glucose-Stimulated Insulin Secretion 
Molecular and Cellular Biology  2005;25(12):4969-4976.
MafA is a transcription factor that binds to the promoter in the insulin gene and has been postulated to regulate insulin transcription in response to serum glucose levels, but there is no current in vivo evidence to support this hypothesis. To analyze the role of MafA in insulin transcription and glucose homeostasis in vivo, we generated MafA-deficient mice. Here we report that MafA mutant mice display intolerance to glucose and develop diabetes mellitus. Detailed analyses revealed that glucose-, arginine-, or KCl-stimulated insulin secretion from pancreatic β cells is severely impaired, although insulin content per se is not significantly affected. MafA-deficient mice also display age-dependent pancreatic islet abnormalities. Further analysis revealed that insulin 1, insulin 2, Pdx1, Beta2, and Glut-2 transcripts are diminished in MafA-deficient mice. These results show that MafA is a key regulator of glucose-stimulated insulin secretion in vivo.
PMCID: PMC1140590  PMID: 15923615

Results 1-9 (9)