scholarly journals Role of Cdx-2 in insulin and proglucagon gene expression: a study using the RIN-1056A cell line with an inducible gene expression system

2005 ◽  
Vol 186 (1) ◽  
pp. 179-192 ◽  
Author(s):  
Yi Zhao ◽  
Tao Liu ◽  
Nina Zhang ◽  
Fenghua Yi ◽  
Qinghua Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Expression System ◽  
Insulin Gene ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Expressions ◽  
Insulin Gene Expression

Although the homeobox gene Cdx-2 was initially isolated from the pancreatic β cell line HIT-T15, no examination of its role in regulating endogenous insulin gene expression has been reported. To explore further the role of Cdx-2 in regulating both insulin and proglucagon gene expression, we established an ecdysone-inducible Cdx-2 expression system. This report describes a study using the rat insulinoma cell line RIN-1056A, which abundantly expresses both insulin and proglucagon (glu), and relatively high amounts of endogenous Cdx-2. Following the introduction of the inducible Cdx-2 expression system into this cell line and the antibiotic selection procedure, we obtained novel cell lines that displayed dramatically reduced expression of endogenous Cdx-2, in the absence of the inducer. These novel cell lines did not express detectable amounts of glu mRNA or the glucagon hormone, while their insulin expression was not substantially affected. In the presence of the inducer, however, transfected Cdx-2 expression was dramatically increased, accompanied by stimulation of endogenous Cdx-2 expression. More importantly, activated Cdx-2 expression was accompanied by elevated insulin mRNA expression, and insulin synthesis. Cdx-2 bound to the insulin gene promoter enhancer elements, and stimulated the expression of a luciferase reporter gene driven by these enhancer elements. Furthermore, Cdx-2 and insulin gene expressions in the wild-type RIN-1056A cells were stimulated by forskolin treatment, and forskolin-mediated activation on insulin gene expression was attenuated in the absence of Cdx-2. We suggest that Cdx-2 may mediate the second messenger cAMP in regulating insulin gene transcription.

scholarly journals Differentially expressed Maf family transcription factors, c-Maf and MafA, activate glucagon and insulin gene expression in pancreatic islet alpha- and beta-cells

2004 ◽  
Vol 32 (1) ◽  
pp. 9-20 ◽  
Author(s):  
K Kataoka ◽  
S Shioda ◽  
K Ando ◽  
K Sakagami ◽  
H Handa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cell Line ◽  
Beta Cells ◽  
Cell Lines ◽  
Promoter Activity ◽  
Insulin Gene ◽  
Insulinoma Cell ◽  
Insulin Gene Expression ◽  
Glucagon And Insulin

A basic-leucine zipper transcription factor, MafA, was recently identified as one of the most important transactivators of insulin gene expression. This protein controls the glucose-regulated and pancreatic beta-cell-specific expression of the insulin gene through a cis-regulatory element called RIPE3b/MARE (Maf-recognition element). Here, we show that MafA expression is restricted to beta-cells of pancreatic islets in vivo and in insulinoma cell lines. We also demonstrate that c-Maf, another member of the Maf family of transcription factors, is expressed in islet alpha-cells and in a glucagonoma cell line (alphaTC1), but not in gamma- and delta-cells. An insulinoma cell line, betaTC6, also expressed c-Maf, albeit at a low level. Chromatin immunoprecipitation assays demonstrated that Maf proteins associate with insulin and glucagon promoters in beta- and alpha-cell lines, respectively. c-Maf protein stimulated glucagon promoter activity in a transient luciferase assay, and activation of the glucagon promoter by c-Maf was more efficient than by the other alpha-cell-enriched transcription factors, Cdx2, Pax6, and Isl-1. Furthermore, inhibition of c-Maf expression in alphaTC1 cells by specific short hairpin RNA resulted in marked reduction of the glucagon promoter activity. Thus, c-Maf and MafA are differentially expressed in alpha- and beta-cells where they regulate glucagon and insulin gene expression, respectively.

microRNA-422a Inhibits DCC Expression in a Manner Dependent on SNP rs12607853

2020 ◽  
Vol 160 (2) ◽  
pp. 63-71
Author(s):  
Yunxiao Li ◽  
Xugang Shi ◽  
Xintong Cai ◽  
Yongsheng Zhu ◽  
Yuanyuan Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Opioid Addiction ◽  
Heroin Addiction ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Protein Levels ◽  
Gene Assay ◽  
New Biomarkers ◽  
And Function

DCC netrin 1 receptor (DCC) affects the structure and function of the dopamine circuitry, which in turn affects the susceptibility to developing addiction. In a previous study, we found that single nucleotide polymorphism (SNP) rs12607853 in the 3′ untranslated region (3′-UTR) of DCC was significantly associated with heroin addiction. In the current study, we first used bioinformatics prediction to identify the DCC rs12607853 C allele as a potential hsa-miR-422a and hsa-miR-378c target site. We then used vector construction and dual-luciferase reporter assays to investigate the targeting relationship of DCC rs12607853 with hsa-miR-422a and hsa-miR-378c. The dual-luciferase reporter gene assay confirmed that the C allele of rs12607853 in combination with hsa-miR-422a led to repressed dual-luciferase gene expression. Moreover, gene expression assays disclosed that hsa-miR-422a inhibited DCC expression at both the mRNA and protein levels. We also found that morphine inhibited the expression of hsa-miR-422a but increased the expression of DCC mRNA, and this change in the expression of hsa-miR-422a could not be reversed by naloxone, which suggested that the role of DCC in opioid addiction might be regulated by hsa-miR-422a. In summary, this study improves our understanding of the role of hsa-miR-422a and identifies the genetic basis of rs12607853, which might contribute to the discovery of new biomarkers or therapeutic targets for opioid addiction.

scholarly journals MiR-624-5p enhances cell resistance against cisplatin via PDGFRA/Stat3/PI3K axis in ovarian cancer

2020 ◽  
Vol 19 (4) ◽  
pp. 691-698
Author(s):  
Lin I-Ju ◽  
Tian YongJie
Keyword(s):  
Ovarian Cancer ◽  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Clinical Stage ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay

Purpose: The purpose of this study was to evaluate the role of miR-624-5p in ovarian cancer.Methods: MiR-624-5p expression in ovarian cancer {OC) cell lines and normal cells (NCs) was evaluated and compared the differential miR-624-5p in OC A2780 cells and cisplatin-resistant OC cell line (A2780/DDP). CCK-8 was used to evaluate changes in cell viability of the A2780 and A2780/DDP cell lines as well as silenced miR-624-5p. Western Blot examined the Stat3 and phosphorylated Pi3k. The binding between PDGFRA and miR-624-5p was predicted on Targetscan and verified through Luciferase Reporter Assay. The role of PDGFRA in A2780/DDP by overexpressing PDGFRA was evaluated by RT-qPCR and CCK-8 assays. RT-qPCR assay also measured miR-624-5p expression responsive to different dosages of cisplatin and CCK8 examined viability levels correspondingly. In addition, the interplay of PDGFRA and miR-624-5p by combined downregulation of both miR-624-5pand PDGFRA were evaluated.Results: OC cells had higher miR-624-5p expression than NCs but lower compared to cisplatinresistant A2780/DDP cells. A2780/DDP cells had higher viability than OC cell line A2780. Stat3 and phosphorylated PI3K were activated in A2780/DDP cells. Silencing miR-624-5p led to lower viability inA2780/DDP cells. miR-624-5p expression dropped as the cisplatin concentration increased, resulting in decreasing viability respectively. Luciferase Reporter assay validated the binding of miR-624-5p and PDGFRA in A2780/DDP cells. Overexpressed PDGFRA induced lower cell viability in A2780/DDP cells. Downregulation of PDGFRA partially restored the lowered viability and inhibited Stat3 as well as phosphorylated Pi3k induced by miR-624-5p inhibitor.Conclusion: MiR-624-5p could add to the cellular resistance to cisplatin in OC in-vitro model, which indicated that it might help unveil the mystery of drug-resistance in clinical stage of ovarian cancer. Keywords: MiR-624-5p, resistance, cisplatin, PDGFRA/Stat3/PI3K, ovarian cancer

Role of the Mitogen-Activated Protein Kinases in Cytokine-Mediated Inhibition of Insulin Gene Expression

2006 ◽  
Vol 54 (3) ◽  
pp. 132-142 ◽  
Author(s):  
Catherine V.T. Chin-Chance ◽  
Marsha V. Newman ◽  
Amy Aronovitz ◽  
Herman Blomeier ◽  
Jessica Kruger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinases ◽  
Insulin Gene ◽  
Mitogen Activated Protein Kinases ◽  
Mitogen Activated Protein ◽  
Insulin Gene Expression

Transcriptional regulation of the cystathionine-β-synthase gene in Down syndrome and non–Down syndrome megakaryocytic leukemia cell lines

Blood ◽  
2003 ◽  
Vol 101 (4) ◽  
pp. 1551-1557 ◽  
Author(s):  
Yubin Ge ◽  
Tanya L. Jensen ◽  
Larry H. Matherly ◽  
Jeffrey W. Taub
Keyword(s):  
Transcriptional Regulation ◽  
Down Syndrome ◽  
Cell Line ◽  
Cell Lines ◽  
Promoter Activity ◽  
Leukemia Cell ◽  
Chromosome 21 ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Nuclear Extracts

Children with Down syndrome (DS) with acute myeloid leukemia (AML) have significantly higher event-free survival rates compared to those with non-DS AML, linked to greater cytosine arabinoside (ara-C) sensitivity and higher transcript levels of the chromosome 21–localized gene, cystathionine-β-synthase(CBS), in DS myeloblasts. In this study, we examined the transcriptional regulation of the CBS gene in the DS megakaryocytic leukemia (AMkL) cell line, CMK, characterized by significantly higher CBS transcripts compared with the non-DS AMkL cell line, CMS. Rapid amplification of 5′-cDNA ends (5′-RACE) analysis demonstrated exclusive use of the CBS−1b promoter in the cell lines, and transient transfections with the full-length CBS −1b luciferase reporter gene construct showed 40-fold greater promoter activity in the CMK than CMS cells. Electrophoretic mobility shift assays showed enhanced binding of the transcription factors Sp1/Sp3 to 2 GC/GT-box elements (GC-f and GT-d) in the upstream regions of the CBS −1b promoter in CMK nuclear extracts and undetectable binding in CMS cells. Mutation of the GC-f– or GT-d–binding site resulted in an approximately 90% decrease of theCBS −1b promoter activity in transient transfections of CMK cells. Chromatin immunoprecipitation assays confirmed in vivo binding of Sp3, USF-1, and nuclear factor YA (NF-YA) to theCBS −1b promoter region in chromatin extracts of CMK and CMS cells. Decreased binding of Sp1/Sp3 in CMK nuclear extracts following treatment with calf alkaline phosphatase suggested a role for phosphorylation of Sp1/Sp3 in regulating CBS promoter activity and in the differential CBS expression between CMK and CMS cells. The results of this study with clinically relevant cell line models suggest potential mechanisms for disparate patterns ofCBS gene expression in DS and non-DS myeloblasts and may, in part, explain the greater sensitivity to chemotherapy shown by patients with DS AML.

scholarly journals SUN-267 Dissecting Type 2 CRH Receptor Signaling Characteristics in the Hypothalamic Cell Line MHYPOA-2/30

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Viridiana Alcantara-Alonso ◽  
Patricia de Gortari ◽  
Robert Dallmann ◽  
Dimitris Grammatopoulos
Keyword(s):  
Gene Expression ◽  
Plasma Membrane ◽  
Protein Expression ◽  
Cell Line ◽  
Cellular Localization ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Luciferase Reporter Gene

Abstract The stress peptides coticotropin-releasing hormone (CRH) and urocortins (Ucns) exert anorectic effects acting mainly through the type 2 CRH receptor (CRH-R2) in the hypothalamus. Impairment of CRH-R2 signaling in chronically stressed rats has been linked with the development of hyperphagia (Alcantara-Alonso et al. Neuropeptides, 2017) however the exact mechanisms and molecular defects are unknown. In the present study we used the mHypoA-2/30, a hypothalamic immortalized cell line derived from adult mice (Belsham et al. FASEB J, 2009) to further explore the signaling molecules mediating the anorexigenic effect of the CRH-R2 cognate agonist urocortin 2 (Ucn2). Specifically, we investigated mRNA, protein expression and cellular localization of CRH-R2 in the mHypoA-2/30 neurons. Additionally, we examined the effects of Ucn2 on the phosphorylation of CREB and AMPK, as well as its transcriptional effects on genes of feeding-related peptides and molecules involved in modulation of circadian rhythms. Both CRH-R2 mRNA and protein expression were detected in mHypoA-2/30; indirect immunoflourescence experiments using a specific CRH-R2 antibody demonstrated widespread localization in the plasma membrane and cytoplasm. Moreover, the receptor sub-cellular localization was redistributed in response to activation by Ucn2 (100 nM), as the plasma membrane immunofluorescent signal was decreased after 4h of agonist treatment, suggesting CRH-R2 homologous internalization. We also observed a 50% increase in the phosphorylation of CREB associated with a concomitant decrease in AMPK phosphorylation after 30 min of Ucn2 treatment. Among the panel of hypothalamic genes analyzed, we identified after 24h of Ucn2 treatment increases in the gene expression of the anorexigenic peptides neurotensin and proopiomelanocortin. Interestingly, sustained CRH-R2 activation also led to an increase in the mRNA levels of Aryl Hydrocarbon Receptor Nuclear Translocator Like (ARNTL), a protein involved in the control of circadian rhythm. A luciferase reporter gene analysis of ARNTL showed that the mHypoA-2/30 cells also exhibit circadian patterns of expression and that the treatment with Ucn2 enhanced circadian amplitude of ARNTL reporter on these cells, which in turn may be involved in glucocorticoid release in circadian cycles and stimulating appetite during the activity phase of the animals. In conclusion, we found that the mHypoA-2/30 cell line expresses endogenous functional CRH-R2 that is linked to downstream regulation of anorexigenic gene expression. This cell line appears to be a useful in vitro tool to study hypothalamic CRH-R2 signaling machinery involved in central control of food intake and circadian cycles.

scholarly journals Prolactin induction of insulin gene expression: the roles of glucose and glucose transporter-2

2000 ◽  
Vol 164 (3) ◽  
pp. 277-286 ◽  
Author(s):  
A Petryk ◽  
D Fleenor ◽  
P Driscoll ◽  
M Freemark
Keyword(s):  
Gene Expression ◽  
Beta Cell ◽  
Gene Transcription ◽  
Glucose Transporter ◽  
Insulin Gene ◽  
Luciferase Reporter ◽  
Insulin Production ◽  
Glucose Transporter 2 ◽  
Insulin Gene Expression ◽  
Insulin Gene Transcription

Previous studies have shown that lactogenic hormones stimulate beta-cell proliferation and insulin production in pancreatic islets. However, all such studies have been conducted in cells incubated in medium containing glucose. Since glucose independently stimulates beta-cell replication and insulin production, it is unclear whether the effects of prolactin (PRL) on insulin gene expression are exerted directly or through the uptake and/or metabolism of glucose. We examined the interactions between glucose and PRL in the regulation of insulin gene transcription and the expression of glucose transporter-2 (glut-2) and glucokinase mRNAs in rat insulinoma (INS-1) cells. In the presence of 5.5 mM glucose, the levels of preproinsulin and glut-2 mRNAs in PRL-treated cells exceeded the levels in control cells (1.7-fold, P<0.05 and 2-fold, P<0.05 respectively). The maximal effects of PRL were noted at 24-48 h of incubation. PRL had no effect on the levels of glucokinase mRNA. The higher levels of glut-2 mRNA were accompanied by an increase in the number of cellular glucose transporters, as demonstrated by a 1. 4- to 2.4-fold increase in the uptake of 2-deoxy-d-[(3)H]glucose in PRL-treated INS-1 cells (P<0.001). These findings suggested that the insulinotropic effect of PRL is mediated, in part, by induction of glucose transport and/or glucose metabolism. Nevertheless, even in the absence of glucose, PRL stimulated increases in the levels of preproinsulin mRNA (3.4-fold higher than controls, P<0.0001) and glut-2 mRNA (2-fold higher than controls, P<0.01). These observations suggested that PRL exerts glucose-independent as well as glucose-dependent effects on insulin gene expression. Support for this hypothesis was provided by studies of insulin gene transcription using INS-1 cells transfected with a plasmid containing the rat insulin 1 promoter linked to a luciferase reporter gene. Glucose and PRL, alone and in combination, stimulated increases in cellular luciferase activity. The relative potencies of glucose (5.5 mM) alone, PRL alone, and glucose plus PRL in combination were 2.2 (P<0.001), 3.4 (P<0.01), and 7.9 (P<0.0001) respectively. Our findings suggest that glucose and PRL act synergistically to induce insulin gene transcription.

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