ognizant Communication Corporation



Gene Expression, Vol. 11, pp. 55-75
1052-2166/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

HGF-, EGF, and Dexamethasone-Induced Gene Expression Patterns During Formation of Tissue in Hepatic Organoid Cultures

George K. Michalopoulos, William C. Bowen, Karen Mulè, and Jianhua Luo

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261

Corticosteroids, hepatocyte growth factor (HGF), and epidermal growth factor (EGF) play important roles in hepatic biology. We have previously shown that these molecules are required for formation of tissue with specific histology in complex organoid cultures. Dexamethasone suppresses growth and induces hepatocyte maturation; HGF and EGF are needed for formation of the nonepithelial elements. All three are needed for formation of the biliary epithelium. The gene expression patterns by which corticosteroids, HGF, and EGF mediate their effects in hepatic tissue formation are distinct. These patterns affect many gene families and are described in detail. In terms of main findings, dexamethasone induces expression of both HNF4 and C/EBPa, essential transcription factors for hepatocyte differentiation. It suppresses hepatocyte growth by suppressing many molecules associated with growth in liver and other tissues, including IL-6, CXC-chemokine receptor, amphiregulin, COX-2, HIF, etc. HGF and EGF induce all members of the TGF-b family. They also induced multiple CNS-related genes, probably associated with stellate cells. Dexamethasone, as well as HGF and EGF, induces expression of HNF6-b, associated with biliary epithelium formation. Combined addition of all three molecules is associated with mature histology in which hepatocyte and biliary lineages are separate and HNF4 is expressed only in hepatocyte nuclei. In conclusion, the results provide new and surprising information on the gene expression alterations by which corticosteroids, HGF, and EGF exert their effects on formation of hepatic tissue. The results underscore the usefulness of the organoid cultures for generating information on histogenesis, which cannot be obtained by other culture or whole animal models.

Key words: Gene arrays; Three-dimensional cultures; Hepatocytes; Biliary cells; Growth regulation

Address correspondence to George K. Michalopoulos, M.D., Ph.D., Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. Tel: (412) 648-1040; Fax: (412) 648-9744; E-mail: michalopoulosgk@msx.upmc.edu

Gene Expression, Vol. 11, pp. 77-83
1052-2166/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Alteration of Gene Expressions by the Overexpression of Mitochondrial Phospholipid Hydroperoxide Glutathione Peroxidase (mtPHGPx)

Jun Kitahara,1 Nobuyoshi Chiba,3 Hikaru Sakamoto,1 and Yasuhito Nakagawa1,2

1Radioisotope Research Laboratory, and 2Department of Hygiene, Pharmaceutical Sciences Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
3Japan Energy Corporation, 3-17-35 Niizo-Minami, Toda-shi, Saitama 335, Japan

To determine the effect on gene expression of trace levels of reactive oxygen species from mitochondria, we used the mRNA differential display technique to compare gene expression in two cell lines: M15, which overexpresses mitochondrial phospholipid hydroperoxide glutathione peroxidase (mtPHGPx), in rat basophilic leukemia RBL-2H3 cells, and a control cell line, S1. We isolated 27 differentially expressed genes, including 10 previously unreported sequences. These genes included cytoskeletal proteins (b-tubulin, nonmuscle myosin alkali light chain, and vimentin), growth or proliferation regulators [growth differentiation factor 1 (Gdf-1), Rap1a, and inhibitor of growth 3 (Ing3)], and others. Although the expression of most of the isolated genes did not respond to ROS (hydrogen peroxide) or antioxidant (pyrolidine dithiocarbamate) treatment, the expression of Gdf-1 was downregulated by hydrogen peroxide treatment. Thus, low levels of ROS produced in mitochondria during normal cellular metabolism can modulate gene expression.

Key words: Phospholipid hydroperoxide glutathione peroxidase (PHGPx); Mitochondria; Reactive oxygen species (ROS); Differential display

Address correspondence to Yasuhito Nakagawa, Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Tel: +81-3-3444-6161, ext. 3336; Fax: +81-3-3444-4944; E-mail: nakagaway@pharm.kitasato-u.ac.jp

Gene Expression, Vol. 11, pp. 85-94
1052-2166/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Characterization of adapt33, a Stress-Inducible Riboregulator

Yanhong Wang,1 Kelvin J. A. Davies,2 J. Andres Melendez,1 and Dana R. Crawford1

1Center for Immunology and Microbial Disease, The Albany Medical College, Albany, NY 12208
2Ethel Percy Andrus Gerontology Center and Division of Molecular Biology, University of Southern California, Los Angeles, CA

We have identified adapt33 as a multiple stress-responsive gene that is induced under conditions of a cytoprotective "adaptive response." adapt33 RNA does not contain any appreciable open reading frame nor produce a protein product and is therefore classified as a stress-inducible riboregulator. Although a number of oxidant stress-modulated, protein-encoding genes have been reported and characterized, very few stress-inducible riboregulator RNAs are known. Here we extend previous studies toward understanding the underlying regulation of expression and function of this rare mammalian riboregulator. mRNA stability and transcription studies determined that adapt33 induction by hydrogen peroxide is at the mRNA stability level, and that adapt33 has a very short half-life. Surprisingly, adapt33 mRNA also exhibits altered electrophoretic migration in response to both hydrogen peroxide and cis-platinum treatment. Although no transcriptional modulation in response to hydrogen peroxide was observed, fusion promoter constructs revealed that adapt33 has an unusually strong promoter that is active in both hamster and human cells. Analysis of expression following the stimulation of apoptosis with hydrogen peroxide and staurosporine revealed a strong correlation with apoptosis, suggesting a possible novel, noncoding RNA component of the apoptotic mechanism. We conclude that adapt33 is a stress-inducible, apoptosis-associated RNA with unique structural and gene promoter characteristics.

Key words: Riboregulator; Adaptive response; Gene expression; Hydrogen peroxide; Oxidative stress; Hamster fibroblasts

Address correspondence to Dana R. Crawford, Ph.D., Center for Immunology and Microbial Disease, The Albany Medical College, Albany, NY 12208. Tel: (518) 262-6652; Fax: (518) 262-5689; E-mail: crawfod@mail.amc.edu

Gene Expression, Vol. 11, pp. 95-104
1052-2166/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
The First Intron of the Human Osteopontin Gene Contains a C/EBP-Beta-Responsive Enhancer

Francesca Giacopelli,1 Nadia Rosatto,1 Maria Teresa Divizia,1 Roberto Cusano,1 Gianluca Caridi,3 and Roberto Ravazzolo1,2

1Laboratory of Molecular Genetics, G. Gaslini Institute, Largo G. Gaslini 5, 16148 Genova, Italy
2Department of Pediatrics and CEBR, University of Genova, Italy
3Laboratory of Nephrology, G. Gaslini Institute, Genova, Italy

The osteopontin (OPN) protein is found expressed at high level in several processes including fibrotic evolution of organ injuries, tumorigenesis, and immune response. The molecular mechanisms that underly overexpression, especially at the transcriptional level, have been only partially clarified. Therefore, this study was undertaken in search for additional DNA elements in the regulatory regions of the OPN gene and cognate transcription factors. Our results on the region upstream of the transcription start site confirmed that essential elements are located within the first 100 bp. Analysis of the sequence that includes the first untranslated exon and first intron revealed that it could enhance the promoter activity. Experiments of transfection of constructs containing different fragments of this sequence showed that most of the enhancer activity was confined in the terminal 30-bp tract of the first intron, although it was not functioning in a myofibroblast cell line. DNA/protein binding assays and cotransfection experiments showed that the C/EBP-beta transcription factor was able to bind a recognition sequence in this 30-bp segment. We found a bi-allelic sequence polymorphism at +245 in the first intron, which did not show a significant functional effect, but is a useful tool for future association studies.

Key words: Osteopontin; Transcription; Enhancer; C/EBP-beta

Address correspondence to Prof. Roberto Ravazzolo, Laboratory of Molecular Genetics, Istituto Giannina Gaslini, Largo G. Gaslini 5, 16148 Genova, Italy. Tel: +39-10-5636400; Fax: +39-10-3779797; E-mail: rravazzo@unige.it

Gene Expression, Vol. 11, pp. 105-116
1052-2166/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Degenerate Suppression PCR Identifies the b2-Adrenergic Receptor as Upregulated by Neuronal Differentiation

Jan Lewerenz, Frank Leypoldt, and Axel Methner

Research Group Protective Signaling, Zentrum für Molekulare Neurobiologie and Department of Neurology, University Hospital Hamburg, Martinistr. 52, D-20246 Hamburg, Germany

Communication between cells is necessary for the functioning of a multicellular organism. Cells process a large amount of information through G-protein-coupled receptors, and activation of this receptor class has been implicated in neuronal differentiation. In this study, we used a method based on PCR with degenerated primers to identify G-protein-coupled receptors regulated by retinoic acid-induced differentiation of the human teratocarcinoma cell line NTera-2/D1. Subtracted cDNA libraries and control cDNA served as templates in half-sided PCR with a forward degenerate primer based on a conserved sequence from human serotonergic, adrenergic, and dopaminergic receptors and reverse primers on adaptors with long terminal repeats commonly employed in subtractive suppression hybridization. We developed conditions to amplify G-protein-coupled receptors from adaptor-ligated cDNA and found the b2-adrenergic receptor to be upregulated fourfold. This seems to be physiologically relevant, as it could also be shown in rat primary cortical cultures maturing in vitro. The method presented here makes use of the otherwise unused control cDNA from subtractive suppression hybridization experiments and could be easily adapted to other gene families.

Key words: NT2 cells; Neuronal differentiation; b2-Adrenergic receptor; Subtractive suppression hybridization; Degenerate PCR

Address correspondence to Axel Methner, Research Group Protective Signaling, Zentrum für Molekulare Neurobiologie Hamburg and Department of Neurology, University Hospital Hamburg, Martinistr. 52, D-20246 Hamburg, Germany. Tel: +49 40 4 28 03 66 26; Fax: +49 40 4 28 03 51 01; E-mail: methner@uke.uni-hamburg.de