Pancreatic Duodenal Homeobox Factor-1 Research Articles

Anti-oxidative and anti-apoptotic effects and molecular mechanisms of catalpol against H_2O_2-induced oxidative damage in pancreatic β cells (INS-1 cells)

The present study investigated the anti-oxidative and anti-apoptotic effects and molecular mechanisms of catalpol on the H_2O_2-induced pancreatic β-cells(INS-1 cells).The oxidative damage model of INS-1 cells was induced and optimized by the stimulation of H_2O_2 of different concentrations for different time.CCK-8 assay was used to detect cell viability after catalpol intervention(1, 5, 10, 20, 40, 80, and 160 μmol·L~(-1)) for 24 h.Intracellular reactive oxygen species(ROS), superoxide dismutase(SOD), and lipid peroxide malondialdehyde(MDA) were measured by DCFH-DA fluorescent probe, WST-1, and TBA respectively.Moreover, the apo-ptotic effect was detected by AO-EB and Annexin V-FITC/PI staining.In addition, the protein expression levels were detected by Wes-tern blot, and intracellular insulin concentration was measured by ELISA.The results showed that the oxidative damage model of INS-1 cells was stably induced by 50 μmol·L~(-1) H_2O_2 treatment for 2 h, and catalpol at 1-80 μmol·L~(-1) did not affect cell viability of INS-1 cells.Compared with the conditions in the model group, 1, 5, and 10 μmol·L~(-1) catalpol intervention for 2 h could protect INS-1 cells from oxidative damage(P<0.001), reduce ROS and MDA, increase SOD, and inhibit excessive cell apoptosis.Moreover, 1, 5, and 10 μmol·L~(-1) catalpol could also up-regulate the phosphorylation of nuclear transcription factor NF-E2 related factors, negatively regulate Kelch-like ECH-associated protein 1(Keap1), phosphorylation of extracellular signal-regulated kinase(ERK), and heme oxyge-nase 1(HO-1), and promote the protein expression of pancreatic-duodenal homeobox factor-1(PDX-1) and glucose transporter 2(GLUT2).In addition, 1, 5, and 10 μmol·L~(-1) catalpol increased insulin secretion of INS-1 cells under oxidative damage in the high-glucose culture medium, indicating function recovery of pancreatic β cells.PDX-1 is a key nuclear transcription factor of pancreatic β cell function that directly regulates GLUT2 and insulin synthesis, and affects glucose homeostasis.In conclusion, catalpol can reduce the oxidative damage and apoptosis of INS-1 cells, activate antioxidant pathway, protect the function of pancreatic β cells, and improve insulin synthesis and secretion.

Improving effect of cordycepin on insulin synthesis and secretion in normal and oxidative-damaged INS-1 cells

Diabetes mellitus (DM) has recently become one of the major diseases that have received attention. Cordycepin (molecular formula: C10H13N5O3), is one of the major bioactive components of Cordyceps militaris, decreases blood glucose levels. In this study, the effect and mechanism of cordycepin in normal and oxidative-damaged INS-1 cells were explored by using cell and molecular biology methods. Results showed that cordycepin could enhance insulin synthesis and secretion. The mechanism is possibly related to the elevated ATP content induced membrane depolarisation and increased Ca2+ concentration. At the genetic level, cordycepin upregulated the mRNA level of insulin, pancreatic duodenal homeobox factor-1 (PDX-1) and glucose transporter 1 (GLUT1). At the protein level, cordycepin promoted the expression of PDX-1, GLUT1, serine threonine kinase (Akt) and phosphorylated Akt (P-Akt). These effects may also contribute to the enhancement of insulin synthesis and secretion. Further analysis revealed that cordycepin protected against H2O2-induced damage on INS-1 cells and improved their viability and insulin synthesis/secretion. This effect should be attributed to the reduced intracellular reactive oxygen species (ROS), enhanced mitochondrial membrane potential (MMP), increased activity of superoxide dismutase (SOD) and upregulated genetic and protein expression of catalase (CAT), PDX-1, GLUT1 and P-Akt. In conclusion, cordycepin promotes insulin synthesis and secretion in normal islet β cells and improves this function in oxidative-damaged islet β cells. Given that islet β cells are vulnerable to oxidative stress, the improving effect of cordycepin on the antioxidant capacity and insulin synthesis/secretion of INS-1 cells may be an important mechanism for its hypoglycaemic effect.

Pancreatic Duodenal Homeobox Factor-1 and Neurogenin-3 Serum Expression in Gestational Diabetes

Objectives: Pancreatic duodenal homeobox factor-1 (PDX-1) and neurogenin-3 (NGN-3) are progenitor cell markers in the pancreas. The aim was to compare their serum levels in women with and without gestational diabetes mellitus (GDM). Material and Methods: This prospective, cross-sectional study included two groups: (a) Women with normal gestation and (b) with GDM. PDX-1 and NGN-3 serum expression was determined by qRT-PCR. Student’s t-test or the Mann–Whitney U-test was used to contrast both groups and the Pearson or Spearman correlation was used. A multiple regression was done introducing body mass index and the relative expression of both genes as independent variables and glucose as dependent variable. Statistical significance was tested at P ≤ 0.05 level. Results: Thirty-eight patients (mean age was of 29.00 ± 7.74 years) were included, 22 belonged to the normal pregnancies, and 16 to GDM. Using the ΔΔCt method, the expression fold change for PDX-1 was 0.458 and for NGN-3 it was 0.361. There was a significant positive correlation between the expressions of both genes. The multiple regression was significant for both genes expression and glucose levels in case of having normal weight. Conclusion: PDX-1 and NGN-3 low serum expression could be predictors of higher glucose levels in normal pregnancies.

Pancreatic Duodenal Homeobox Factor-1 (PDX-1) and Neurogenin-3 (NGN-3) in Gestational Diabetes

Pancreatic Duodenal Homeobox Factor-1 (PDX-1) and Neurogenin-3 (NGN-3) in Gestational Diabetes

Circulating Adrenomedullin Is Elevated in Gestational Diabetes and Its Role in Impaired Insulin Production by β-Cells.

Defective pancreatic β-cell adaptation in pregnancy plays an important role in the pathophysiology of gestational diabetes mellitus (GDM), but the molecular basis remains unclear. Objectives of this study were to determine if circulating levels of adrenomedullin (ADM) in women with GDM are elevated and to assess the effects of ADM on insulin synthesis and secretion by human pancreatic β-cells. A stable gene product of ADM precursor, midregional pro-adrenomedullin(MR-proADM), was measured in plasma of pregnant women with normal glucose tolerance (NGT, n = 10) or GDM (n = 11). The β-Lox5 cell line, derived from human pancreatic β-cells, was transduced with homeodomain transcription factor pancreatic-duodenal homeobox (PDX) factor 1 (PDX1) encoding lentiviral vector and treated with different doses of ADM. mRNA for insulin, ADM, and its receptor components in β-Lox5 cells and insulin in media were measured. Plasma MR-proADM levels were significantly higher in GDM compared with patients with NGT. Pancreatic β-Lox5 cells express mRNA for insulin, ADM, and its receptor components. PDX1 transduction and cell-cell contact synergistically promote β-Lox5 cells insulin mRNA and secretion. Furthermore, ADM dose-dependently inhibited mRNA and secretion of insulin in β-Lox5 cell aggregates. These inhibitory effects were blocked by ADM antagonist ADM22-52, cAMP-dependent protein kinase A inhibitor KT5720, and Erk inhibitor PD98059, but not by PI-3K the inhibitor wortmannin. Circulating ADM concentrations were elevated in pregnant women with GDM. ADM suppresses insulin synthesis and secretion by pancreatic β-cells in vitro. Thus, increased circulating ADM may contribute to the defective adaptation of β-cells in diabetic pregnancies, and blockade of ADM actions with its antagonists may improve β-cell functions.

Basement Membrane Extract Preserves Islet Viability and Activity In Vitro by Up-Regulating α3 Integrin and Its Signal

Survival of transplanted islets is limited partly because of the disruption of the islet basement membrane (BM) occurring during isolation. We hypothesized that the embedment of BM extract (BME) could induce a viable cell mass and prolong islet functionality before transplantation. A special reconstituted BME that solidifies into a gel at 37°C was used to embed isolated islets in this study. The strategy was used to re-establish the interaction between the islets and peri-islet BM. Islets embedded in BME showed lower caspase-3 levels and higher Akt activity than those in suspension. Moreover, we found for the first time that the expression of α3 integrin and focal adhesion kinase (FAK) and FAK activity was up-regulated in islets after BME embedment. The reverse effect was observed on islet apoptosis when islets rescued from a 24-hour suspension culture were embedded in BME for the next 24 hours. In addition, expression of pancreatic duodenal homeobox factor-1 and phospho-extracellular signal-regulated kinase 1/2 was partially preserved, suggesting the positive effect of BME on islet development. These results indicate that BME embedment of islets can up-regulate the expression of α3 integrin and its signal transduction, which may improve islet viability.

Participation of peribiliary glands in biliary tract pathophysiologies

To investigate the roles of peribiliary glands around the bile ducts in the pathophysiology of the biliary tract. The expression of fetal pancreatic markers, pancreatic duodenal homeobox factor 1 (PDX1) and hairy and enhancer of split 1 (HES1) and endodermal stem/progenitor (S/P) cell markers [CD44s, chemokine receptor type 4 (CXCR4), SOX9 and epithelial cell adhesion molecule (EpCAM)] were examined immunohistochemically in 32 normal adult livers (autopsy livers) and 22 hepatolithiatic livers (surgically resected livers). The latter was characterized by the proliferation of the peribiliary glands. Immunohistochemistry was performed using formalin-fixed, paraffin-embedded tissue sections after deparaffinization. Although PDX1 and HES1 were expressed in both the nucleus and cytoplasm of epithelial cells, only nuclear staining was evaluated. SOX9 was expressed in the nucleus, while CD44s, CXCR4 and EpCAM were expressed in the cell membranes. The frequency and extent of the expression of these molecules in the lining epithelia and peribiliary glands were evaluated semi-quantitatively based on the percentage of positive cells: 0, 1+ (focal), 2+ (moderate) and 3+ (extensive). In normal livers, PDX1 was infrequently expressed in the lining epithelia, but was frequently expressed in the peribiliary glands. In contrast, HES1 was frequently expressed in the lining epithelia, but its expression in the peribiliary glands was focal, suggesting that the peribiliary glands retain the potential of differentiation toward the pancreas and the lining epithelia exhibit properties to inhibit such differentiation. This unique combination was also seen in hepatolithiatic livers. The expression of endodermal S/P cell markers varied in the peribiliary glands in normal livers: SOX9 and EpCAM were frequently expressed, CD44s infrequently, and CXCR4 almost not at all. The expression of these markers, particularly CD44s and CXCR4, increased in the peribiliary glands and lining epithelia in hepatolithiatic livers. This increased expression of endodermal S/P cell markers may be related to the increased production of intestinal and gastric mucin and also to the biliary neoplasia associated with the gastric and intestinal phenotypes reported in hepatolithiasis. The unique expression pattern of PDX1 and HES1 and increased expression of endodermal S/P cell markers in the peribiliary glands may be involved in biliary pathophysiologies.

Mediation of glucolipotoxicity in INS-1 rat insulinoma cells by small heterodimer partner interacting leucine zipper protein (SMILE)

Sustained elevations of glucose and free fatty acid concentration have deleterious effects on pancreatic beta cell function. One of the hallmarks of such glucolipotoxicity is a reduction in insulin gene expression, resulting from decreased insulin promoter activity. Sterol regulatory element binding protein-1c (SREBP-1c), a lipogenic transcription factor, is related to the development of beta cell dysfunction caused by elevated concentrations of glucose and free fatty acid. Small heterodimer partner (SHP) interacting leucine zipper protein (SMILE), also known as Zhangfei, is a novel protein which interacts with SHP that mediates glucotoxicity in INS-1 rat insulinoma cells. Treatment of INS-1 cells with high concentrations of glucose and palmitate increased SREBP-1c and SMILE expression, and decreased insulin gene expression. Adenovirus-mediated overexpression of SREBP-1c in INS-1 cells induced SMILE expression. Moreover, adenovirus-mediated overexpression of SMILE (Ad-SMILE) in INS-1 cells impaired glucose-stimulated insulin secretion as well as insulin gene expression. Ad-SMILE overexpression also inhibited the expression of beta-cell enriched transcription factors including pancreatic duodenal homeobox factor-1, beta cell E box transactivator 2 and RIPE3b1/MafA, in INS-1 cells. Finally, in COS-1 cells, expression of SMILE inhibited the insulin promoter activity induced by these same beta-cell enriched transcription factors. These results collectively suggest that SMILE plays an important role in the development of beta cell dysfunction induced by glucolipotoxicity.

AGEs Decrease Insulin Synthesis in Pancreatic β-Cell by Repressing Pdx-1 Protein Expression at the Post-Translational Level

Advanced glycation end products (AGEs) have been implicated in diverse pathological settings of many diabetic complications, and the possible mechanisms have been widely reported. However, the relationship between AGEs and pancreatic β-cell dysfunction is still poorly understood. Recent studies have shown that AGEs can impair β-cell function by inducing apoptosis or decreasing insulin secretion. Our previous research revealed that AGEs could significantly down-regulate insulin transcription and reduce β-cell glucose-stimulated insulin secretion (GSIS). Here, we investigated the possible mechanisms underlying AGE-related suppression of insulin synthesis. In the rat pancreatic β-cell line INS-1, we found that AGEs induced dephosphorylation of Foxo1 and increased its accumulation in the nucleus. The translocation of Foxo1 subsequently inhibited pancreatic-duodenal homeobox factor-1 (Pdx-1) levels in both nuclear and cytoplasmic compartments. We observed that with AGEs treatment, Pdx-1 protein levels decreased after 4 h, but there was no change in the Pdx-1 mRNA level or promoter activity at the same time point; this demonstrated that the decrease in Pdx-1 expression was not regulated at the transcriptional level. In our study, the decrease in Pdx-1 protein level was related to its reduced stability, overexpression of DN-Foxo1 could partially reverse the inhibition of Pdx-1 expression. Pretreatment with AGEs receptor (RAGE) antibody also prevented the AGE-induced diminution of Pdx-1 protein and insulin mRNA expression. In summary, AGEs induced nuclear accumulation of Foxo1; this in turn reduced Pdx-1 expression by decreasing its protein stability, ultimately affecting insulin synthesis.

The role of protein kinase CK2 in the regulation of the insulin production of pancreatic islets

An appropriate regulation of the insulin production and secretion in pancreatic β-cells is necessary for the control of blood glucose homeostasis. The pancreatic duodenal homeobox factor-1 (Pdx-1) is among the various factors and signals which are implicated in the regulation of the insulin synthesis and secretion in the pancreatic β-cells. Recently, we identified Pdx-1 as a substrate for protein kinase CK2. Since CK2 is implicated in the regulation of many different cellular signaling pathways we now asked whether it might also be involved in the regulation of the insulin regulation in β-cells. Here, we show that insulin treatment of β-cells resulted in an elevated CK2 kinase activity. On the other hand down-regulation of CK2 activity by quinalizarin led to an elevated level of insulin. These results demonstrate that CK2 is implicated in the insulin regulation on pancreatic β-cells.

Evidence for Epithelial-Mesenchymal Transition in Adult Human Pancreatic Exocrine Cells

It has been shown that adult pancreatic ductal cells can dedifferentiate and act as pancreatic progenitors. Dedifferentiation of epithelial cells is often associated with the epithelial-mesenchymal transition (EMT). In this study, we investigated the occurrence of EMT in adult human exocrine pancreatic cells both in vitro and in vivo. Cells of exocrine fraction isolated from the pancreas of brain-dead donors were first cultured in suspension for eight days. This led to the formation of spheroids, composed of a principal population of cells with duct-like phenotype. When cultivated in tissue culture-treated flasks, spheroid cells exhibited a proliferative capacity and coexpressed epithelial (cytokeratin7 and cytokeratin19) and mesenchymal (vimentin and alpha-smooth muscle actin) markers as well as marker of progenitor pancreatic cells (pancreatic duodenal homeobox factor-1) and surface markers of mesenchymal stem cells. The switch from E-cadherin to N-cadherin associated with Snail1 expression suggested that these cells underwent EMT. In addition, we showed coexpression of epithelial and mesenchymal markers in ductal cells of one normal adult pancreas and three type 2 diabetic pancreases. Some of the vimentin-positive cells were found to coexpress glucagon or amylase. These results point to the occurrence of EMT, which may take place on dedifferentiation of ductal cells during the regeneration or renewal of human pancreatic tissues.

Neurogenin 3 and Neurogenic Differentiation 1 are Retained in the Cytoplasm of Multiple Endocrine Neoplasia Type 1 Islet and Pancreatic Endocrine Tumor Cells

To investigate if transcription factors involved in pancreatic differentiation and regeneration are present in pancreatic endocrine tumors and if they are differentially expressed in normal pancreas compared with multiple endocrine neoplasia type 1 (MEN1) nontumorous pancreas. The expression of neurogenin 3 (NEUROG3), neurogenic differentiation 1 (NEUROD1), POU class 3 homeobox 4 (POU3F4), pancreatic duodenal homeobox factor 1 (PDX1), ribosomal protein L10 (RPL10), delta-like 1 homolog (Drosophila; DLK1), and menin was analyzed by immunohistochemistry in normal pancreas and pancreatic endocrine tumors from 6 patients with MEN1 and 16 patients with sporadic tumors, as well as pancreatic specimens from Men1 heterozygous and wild type mice. Quantitative polymerase chain reaction was performed in a subset of human tumors. Tumors and MEN1 nontumorous endocrine cells showed a prominent cytoplasmatic NEUROG3 and NEUROD1 expression. These factors were significantly more expressed in the cytoplasm of Men1 heterozygous mouse islet cells compared with wild type islets; the latter showed an exclusively nuclear reactivity. The degree of Pou3f4, Rpl10, and Dlk1 immunoreactivities differed significantly between islets of heterozygous and wild type mice. The expressions of RPL10 and NEUROD1 were prominent in the MEN1 human and heterozygous mouse exocrine pancreas. Insulinomas had significantly higher PDX1 and DLK1 messenger RNA levels compared with other tumor types. Transcription factors involved in pancreatic development show altered expression and subcellular localization in MEN1 nontumorous pancreas and pancreatic endocrine tumors.

MafA and MafB Regulate Pdx1 Transcription through the Area II Control Region in Pancreatic β Cells

Pancreatic-duodenal homeobox factor-1 (Pdx1) is highly enriched in islet β cells and integral to proper cell development and adult function. Of the four conserved 5′-flanking sequence blocks that contribute to transcription in vivo, Area II (mouse base pairs -2153/-1923) represents the only mammalian specific control domain. Here we demonstrate that regulation of β-cell-enriched Pdx1 expression by the MafA and MafB transcription factors is exclusively through Area II. Thus, these factors were found to specifically activate through Area II in cell line transfection-based assays, and MafA, which is uniquely expressed in adult islet β cells was only bound to this region in quantitative chromatin immunoprecipitation studies. MafA and MafB are produced in β cells during development and were both bound to Area II at embryonic day 18.5. Expression of a transgene driven by Pdx1 Areas I and II was also severely compromised during insulin+ cell formation in MafB-/- mice, consistent with the importance of this large Maf in β-cell production and Pdx1 expression. These findings illustrate the significance of large Maf proteins to Pdx1 expression in β cells, and in particular MafB during pancreatic development.

Endoplasmic Reticulum Stress-Induced Activation of Activating Transcription Factor 6 Decreases Insulin Gene Expression via Up-Regulation of Orphan Nuclear Receptor Small Heterodimer Partner

The highly developed endoplasmic reticulum (ER) structure of pancreatic beta-cells is a key factor in beta-cell function. Here we examined whether ER stress-induced activation of activating transcription factor (ATF)-6 impairs insulin gene expression via up-regulation of the orphan nuclear receptor small heterodimer partner (SHP; NR0B2), which has been shown to play a role in beta-cell dysfunction. We examined whether ER stress decreases insulin gene expression, and this process is mediated by ATF6. A small interfering RNA that targeted SHP was used to determine whether the effect of ATF6 on insulin gene expression is mediated by SHP. We also measured the expression level of ATF6 in pancreatic islets in Otsuka Long Evans Tokushima Fatty rats, a rodent model of type 2 diabetes. High glucose concentration (30 mmol/liter glucose) increased ER stress in INS-1 cells. ER stress induced by tunicamycin, thapsigargin, or dithiotreitol decreased insulin gene transcription. ATF6 inhibited insulin promoter activity, whereas X-box binding protein-1 and ATF4 did not. Adenovirus-mediated overexpression of active form of ATF6 in INS-1 cells impaired insulin gene expression and secretion. ATF6 also down-regulated pancreatic duodenal homeobox factor-1 and RIPE3b1/MafA gene expression and repressed the cooperative action of pancreatic duodenal homeobox factor-1, RIPE3b1/MafA, and beta-cell E box transactivator 2 in stimulating insulin transcription. The ATF6-induced suppression of insulin gene expression was associated with up-regulation of SHP gene expression. Finally, we found that expression of ATF6 was increased in the pancreatic islets of diabetic Otsuka Long Evans Tokushima Fatty rats, compared with their lean, nondiabetic counterparts, Long-Evans Tokushima Otsuka rats. Collectively, this study shows that ER stress-induced activation of ATF6 plays an important role in the development of beta-cell dysfunction.

Pancreatic duodenal homeobox factor-1and diabetes mellitus type 2 (Review)

The homeobox domain transcription factor PDX-1 is essential for pancreatic development and for the maintenance of beta-cell function. The participation of pancreatic duodenal homeobox factor-1 (PDX-1) in the transcription of several genes which are essential for glucose sensing and insulin synthesis underlines its key role in beta-cells of the pancreas. PDX-1 binds to the promoter of insulin, glucose transporter 2, and glucokinase and regulates their expression. By protein-protein interaction, PDX-1 acts in concert with other transcription factors or coactivators at the level of the insulin promoter. Ectopic expression of PDX-1 together with other cofactors can re-program cells to behave like beta-cells and produce insulin. This property of PDX-1 opens new strategies for the treatment of diabetes. Little is known about its regulation at the posttranslational level. Here, we report on its DNA-binding activity, the nuclear import and on post-translational modifications such as phosphorylation, glycosylation and sumoylation. Modulation of these post-translational modifications may be an alternate strategy for treating diabetes.

Cell-specific effects of human cytomegalovirus unique region on recombinant protein expression

The major immediate-early (MIE) promoter of human cytomegalovirus (CMV) is widely used to express recombinant proteins in mammalian cells. CMV MIE promoter contains a strong enhancer and an AT-rich unique region (UR). The UR can function as an insulator or a negative element of CMV MIE promoter, depending on the cellular proteins associated with it. To examine the effects of UR on recombinant protein expression in mammalian cells, we constructed two CMV MIE promoter-based expression plasmids for comparison to the conventional CMV MIE promoter by removing or adding UR. Addition of UR enhances transgene expression in HEK293 stable cells while removal of UR increases both transient and stably integrated expression in HeLa cells. Our results further demonstrate that the cell-specific effect of UR depends on the protein levels of UR-binding proteins, pancreatic-duodenal homeobox factor-1, special AT-rich sequence binding protein 1, and CCAAT displacement protein, in these cells. Collectively, these modified CMV expression plasmids can be utilized to improve recombinant protein production in specific mammalian cell lines.

Cellular homeoproteins, SATB1 and CDP, bind to the unique region between the human cytomegalovirus UL127 and major immediate-early genes

An AT-rich region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region (UR). It has been shown that the UR represses activation of transcription from the UL127 promoter and functions as a boundary between the divergent UL127 and MIE genes during human CMV infection [Angulo, A., Kerry, D., Huang, H., Borst, E.M., Razinsky, A., Wu, J., Hobom, U., Messerle, M., Ghazal, P., 2000. Identification of a boundary domain adjacent to the potent human cytomegalovirus enhancer that represses transcription of the divergent UL127 promoter. J. Virol. 74 (6), 2826-2839; Lundquist, C.A., Meier, J.L., Stinski, M.F., 1999. A strong negative transcriptional regulatory region between the human cytomegalovirus UL127 gene and the major immediate-early enhancer. J. Virol. 73 (11), 9039–9052]. A putative forkhead box-like (FOX-like) site, AAATCAATATT, was identified in the UR and found to play a key role in repression of the UL127 promoter in recombinant virus-infected cells [Lashmit, P.E., Lundquist, C.A., Meier, J.L., Stinski, M.F., 2004. Cellular repressor inhibits human cytomegalovirus transcription from the UL127 promoter. J. Virol. 78 (10), 5113–5123]. However, the cellular factors which associate with the UR and FOX-like region remain to be determined. We reported previously that pancreatic-duodenal homeobox factor-1 (PDX1) bound to a 45-bp element located within the UR [Chao, S.H., Harada, J.N., Hyndman, F., Gao, X., Nelson, C.G., Chanda, S.K., Caldwell, J.S., 2004. PDX1, a Cellular Homeoprotein, Binds to and Regulates the Activity of Human Cytomegalovirus Immediate Early Promoter. J. Biol. Chem. 279 (16), 16111–16120]. Here we demonstrate that two additional cellular homeoproteins, special AT-rich sequence binding protein 1 (SATB1) and CCAAT displacement protein (CDP), bind to the human CMV UR in vitro and in vivo. Furthermore, CDP is identified as a FOX-like binding protein and a repressor of the UL127 promoter, while SATB1 has no effect on UL127 expression. Since CDP is known as a transcription repressor and a nuclear matrix-associated region binding protein, CDP may have a role in the regulation of human CMV transcription.

SOX6 Suppresses Cyclin D1 Promoter Activity by Interacting with β-Catenin and Histone Deacetylase 1, and Its Down-regulation Induces Pancreatic β-Cell Proliferation

Sex-determining region Y-box (SOX) 6 negatively regulates glucose-stimulated insulin secretion from beta-cells and is a down-regulated transcription factor in the pancreatic islet cells of hyperinsulinemic obese mice. To determine the contribution of SOX6 to insulin resistance, we analyzed the effects of SOX6 on cell proliferation. Small interfering RNA-mediated attenuation of SOX6 expression stimulated the proliferation of insulinoma INS-1E and NIH-3T3 cells, whereas retroviral overexpression resulted in inhibition of cell growth. Quantitative real time-PCR analysis revealed that the levels of cyclin D1 transcripts were markedly decreased by SOX6 overexpression. Luciferase-reporter assay with beta-catenin showed that SOX6 suppresses cyclin D1 promoter activities. In vitro binding experiments showed that the LZ/Q domain of SOX6 physically interacts with armadillo repeats 1-4 of beta-catenin. Furthermore, chromatin immunoprecipitation assay revealed that increased SOX6 expression significantly reduced the levels of acetylated histones H3 and H4 at the cyclin D1 promoter. By using a histone deacetylase (HDAC) inhibitor and co-immunoprecipitation analysis, we showed that SOX6 suppressed cyclin D1 activities by interacting withbeta-catenin and HDAC1. The data presented suggest that SOX6 may be an important factor in obesity-related insulin resistance.

MafA Expression and Insulin Promoter Activity Are Induced by Nicotinamide and Related Compounds in INS-1 Pancreatic β-Cells

Nicotinamide has been reported to induce differentiation of precursor/stem cells toward a beta-cell phenotype, increase islet regeneration, and enhance insulin biosynthesis. Exposure of INS-1 beta-cells to elevated glucose leads to reduced insulin gene transcription, and this is associated with diminished binding of pancreatic duodenal homeobox factor 1 (PDX-1) and mammalian homologue of avian MafA/l-Maf (MafA). Nicotinamide and other low-potency poly(ADP-ribose) polymerase (PARP) inhibitors were thus tested for their ability to restore insulin promoter activity. The low-potency PARP inhibitors nicotinamide, 3-aminobenzamide, or PD128763 increased expression of a human insulin reporter gene suppressed by elevated glucose. In contrast, the potent PARP-1 inhibitors PJ34 or INO-1001 had no effect on promoter activity. Antioxidants, including N-acetylcysteine, lipoic acid, or quercetin, only minimally induced the insulin promoter. Site-directed mutations of the human insulin promoter mapped the low-potency PARP inhibitor response to the C1 element, which serves as a MafA binding site. INS-1 cells exposed to elevated glucose had markedly reduced MafA protein and mRNA levels. Low-potency PARP inhibitors restored MafA mRNA and protein levels, but they had no affect on PDX-1 protein levels or binding activity. Increased MafA expression by low-potency PARP inhibitors was independent of increased MafA protein or mRNA stability. These data suggest that low-potency PARP inhibitors increase insulin biosynthesis, in part, through a mechanism involving increased MafA gene transcription.

Failure of Transplanted Bone Marrow Cells to Adopt a Pancreatic β-Cell Fate

Recent studies in normal mice have suggested that transplanted bone marrow cells can transdifferentiate into pancreatic beta-cells at relatively high efficiency. Herein, adopting the same and alternative approaches to deliver and fate map-transplanted bone marrow cells in the pancreas of normal as well as diabetic mice, we further investigated the potential of bone marrow transplantation as an alternative approach for beta-cell replacement. In contrast to previous studies, transplanted bone marrow cells expressing green fluorescence protein (GFP) under the control of the mouse insulin promoter failed to express GFP in the pancreas of normal as well as diabetic mice. Although bone marrow cells expressing GFP under the ubiquitously expressed beta-actin promoter efficiently engrafted the pancreas of normal and hyperglycemic mice, virtually all expressed CD45 and Mac-1/Gr-1, demonstrating that they adopt a hematopoietic rather than beta-cell fate, a finding further substantiated by the complete absence of GFP(+) cells expressing insulin and the beta-cell transcription factors pancreatic duodenal homeobox factor-1 and homeodomain protein. Thus, transplanted bone marrow cells demonstrated little, if any, capacity to adopt a beta-cell fate.