- Email: [email protected]
Glucocorticoid regulation of the rat plasminogen activator inhibitor-1 (PAI-1) gene in FTO-2b hepatoma cells is mediated through a complex GRE
80
221
ORAL
COMMUNICATATIONS
111-5:Regulation
of Gene Expression:
CHARACTERIZATION OF A NEW TRANSCRIPTION FACTOR INVOLVED IN HUMAN PAIGENE EXPRESSION. Ding H, Descheemaeker K, Belayew A and Collen D Center for Molecular and Vascular Biology, University of Leuven, Belgium. In transient expression experiments, the proximal promoter of the PAI- gene confers phorbol ester induction onto a linked CAT reporter gene in transfected HeLa and HT1080 ceils (1). Two regulatory elements are involved in this response: box A (-50 to -65 bp) which binds AP-1 and box B (-82 to -65 bp) which interacts with AP-2, SP-1 and a new transcription factor (2) whose DNA binding is suppressed by phosphatase treatment. Scanning of a HeLa cell cDNA expression library with box B as a probe identified a 500 bp cDNA fragment which was fused to the GST gene in the pGEX vector to express a fusion protein in E. Co/i. The fusion protein specifically interacted with box B in electrophoretic mobility shift assays (EMSA). Antibodies raised against the fusion protein
3
interfere with nuclear proteins that form a complex with box B in EMSA. Screenings with the 500 bp fragment yielded a full length cDNA encoding a 114 kDa protein, the sequence of which was not found in the EMBL data bank. It contains regions with homology to nucleotide-binding and helicase domains of “global transcription factors” (3) on the one hand, and “RING-finger” domain of a zinc-finger family (4) on the other hand and in addition several putative protein kinase C phosphorylation sites In order to study the biological activity of this new protein its full length cDNA has been reconstituted and put under the control of the CMV promoter/enhancer. Co-transfection experiments of HeLa and HT1080 cells with the PAI-l-CAT reporter gene and this expression vector are in progress. 1. Descheemaeker et al. J. Biol. Chem. 1992; 267: 15086-l 5091. 2. Descheemaeker et al. Fibrinolysis 1992; 6: 256-262. 3. Tamkun et al. Cell 1992; 68, 561-572. 4. Lovering et al. Proc. Natl. Acad. Sci. USA 1993; 90: 21 12-21 16.
222 GLUCOCORTICOID
REGULATION OF THE RAT PLASMINOGEN ACTIVATOR INHIBITOR-l (PAI-1) GENE IN FTO-2B HEPATOMA CELLS IS MEDIATED THROUGH A COMPLEX GRE CJ Bruzdzinski’ and TD Gelehrte? ‘University of Illinois at Chicago, and *University of Michigan, Ann Arbor, USA Glucocorticoids induce plasminogen activator inhibitor-l (PAI-1) gene expression in several rat hepatoma cell lines, including HTC and FTO-2B cells. This regulation is accomplished at the level of gene transcription. While induction in HTC cells is mediated by a simple GRE, requiring only the glucocorticoid receptor for response, the mechanism in FTO-2B cells is more complex, involving three different regions of the PAI- promoter. To analyze one of these regions, we have created a series of linker scanner mutants which replace only specific sequences within the context of the rat PAI- promoter.
These mutant promoters were fused to the chloramphenicol acetyltransferase gene and transfected into FTO-2B cells. Analysis of CAT expression in response to dexamethasone indicated that a complex GRE located approximately 550 base pairs upstream of the start site of transcription is involved in the response. Mutation of either a GRE 15mer or an adjacent NF-1 site within this composite element abolishes the response; therefore, at least one other protein is necessary for the glucocorticoid response elicited through these sequences. Further analyses of this site and the identity of this protein(s) is currently being investigated.
223
COMPARATIVE INHIBITION OF PAI-I EXPRESSION IN HUMAN ENDOTHELIAL CELLS BY MODIFIED OLIGODEOXYNUCLEOTIDES ‘Cierniewski CS, ‘Babinska A, ‘Swiatkowska M, ‘Wilczvnska M, ‘Okruszek A, ‘Stec WJ ‘Department of Biophysics, Medical University in Lodz, Poland and ‘Center for Molecular and Macromolecular Studies, Lodz, Poland
through an enzymatic activity. To assess the capability that these agents may modulate PAI- mediated fibrinolysis balance we have designed oligodeoxynucleotides antisense to mRNA sequences for the human PAI-I. We have studied the translation of PAI-I mRNA in human endothelial cells (HUVECs) in the presence of anti-sense oligodeoxynucleotides. Results obtained with the unmodified all-oxygen compounds were compared with those obtained when phosphorothioates were used. Concentration of PAI- in conditioned media and HUVECs was determined by (a) ELISA, (b) the activity test with fibrin as a substrate, and (c) by immunoprecipitation after metabolic labelling of cells with [%]methionine. Two phosphorothioate 24-mers were specifically inhibitory. They targeted to the coding and noncoding region of PAI-I mRNA, respectively. The unmodified all-oxygen compounds did not influence PAIexpression in HUVECs. In summary, we present evidence that the expression elf PAI-I in HUVECs can be selectively inhihited by short antisense oligonucleotides
Recently, there has been a growing interest in the potential application of antisense oligonucleotides in the inhibition of cellular and viral gene expression. This approach is based on the assumption that short complementary DNA sequences might interact specifically with mRNA to form double-stranded helices, and so inhibit nuclear processing of the immature mRNA, transport into cytoplasm, or cytoplasmic translation into proteins. Alternatively, interaction of antisense oligonucleotides with nucleic acids may stimulate degradation of the message
221
ORAL
COMMUNICATATIONS
111-5:Regulation
of Gene Expression:
CHARACTERIZATION OF A NEW TRANSCRIPTION FACTOR INVOLVED IN HUMAN PAIGENE EXPRESSION. Ding H, Descheemaeker K, Belayew A and Collen D Center for Molecular and Vascular Biology, University of Leuven, Belgium. In transient expression experiments, the proximal promoter of the PAI- gene confers phorbol ester induction onto a linked CAT reporter gene in transfected HeLa and HT1080 ceils (1). Two regulatory elements are involved in this response: box A (-50 to -65 bp) which binds AP-1 and box B (-82 to -65 bp) which interacts with AP-2, SP-1 and a new transcription factor (2) whose DNA binding is suppressed by phosphatase treatment. Scanning of a HeLa cell cDNA expression library with box B as a probe identified a 500 bp cDNA fragment which was fused to the GST gene in the pGEX vector to express a fusion protein in E. Co/i. The fusion protein specifically interacted with box B in electrophoretic mobility shift assays (EMSA). Antibodies raised against the fusion protein
3
interfere with nuclear proteins that form a complex with box B in EMSA. Screenings with the 500 bp fragment yielded a full length cDNA encoding a 114 kDa protein, the sequence of which was not found in the EMBL data bank. It contains regions with homology to nucleotide-binding and helicase domains of “global transcription factors” (3) on the one hand, and “RING-finger” domain of a zinc-finger family (4) on the other hand and in addition several putative protein kinase C phosphorylation sites In order to study the biological activity of this new protein its full length cDNA has been reconstituted and put under the control of the CMV promoter/enhancer. Co-transfection experiments of HeLa and HT1080 cells with the PAI-l-CAT reporter gene and this expression vector are in progress. 1. Descheemaeker et al. J. Biol. Chem. 1992; 267: 15086-l 5091. 2. Descheemaeker et al. Fibrinolysis 1992; 6: 256-262. 3. Tamkun et al. Cell 1992; 68, 561-572. 4. Lovering et al. Proc. Natl. Acad. Sci. USA 1993; 90: 21 12-21 16.
222 GLUCOCORTICOID
REGULATION OF THE RAT PLASMINOGEN ACTIVATOR INHIBITOR-l (PAI-1) GENE IN FTO-2B HEPATOMA CELLS IS MEDIATED THROUGH A COMPLEX GRE CJ Bruzdzinski’ and TD Gelehrte? ‘University of Illinois at Chicago, and *University of Michigan, Ann Arbor, USA Glucocorticoids induce plasminogen activator inhibitor-l (PAI-1) gene expression in several rat hepatoma cell lines, including HTC and FTO-2B cells. This regulation is accomplished at the level of gene transcription. While induction in HTC cells is mediated by a simple GRE, requiring only the glucocorticoid receptor for response, the mechanism in FTO-2B cells is more complex, involving three different regions of the PAI- promoter. To analyze one of these regions, we have created a series of linker scanner mutants which replace only specific sequences within the context of the rat PAI- promoter.
These mutant promoters were fused to the chloramphenicol acetyltransferase gene and transfected into FTO-2B cells. Analysis of CAT expression in response to dexamethasone indicated that a complex GRE located approximately 550 base pairs upstream of the start site of transcription is involved in the response. Mutation of either a GRE 15mer or an adjacent NF-1 site within this composite element abolishes the response; therefore, at least one other protein is necessary for the glucocorticoid response elicited through these sequences. Further analyses of this site and the identity of this protein(s) is currently being investigated.
223
COMPARATIVE INHIBITION OF PAI-I EXPRESSION IN HUMAN ENDOTHELIAL CELLS BY MODIFIED OLIGODEOXYNUCLEOTIDES ‘Cierniewski CS, ‘Babinska A, ‘Swiatkowska M, ‘Wilczvnska M, ‘Okruszek A, ‘Stec WJ ‘Department of Biophysics, Medical University in Lodz, Poland and ‘Center for Molecular and Macromolecular Studies, Lodz, Poland
through an enzymatic activity. To assess the capability that these agents may modulate PAI- mediated fibrinolysis balance we have designed oligodeoxynucleotides antisense to mRNA sequences for the human PAI-I. We have studied the translation of PAI-I mRNA in human endothelial cells (HUVECs) in the presence of anti-sense oligodeoxynucleotides. Results obtained with the unmodified all-oxygen compounds were compared with those obtained when phosphorothioates were used. Concentration of PAI- in conditioned media and HUVECs was determined by (a) ELISA, (b) the activity test with fibrin as a substrate, and (c) by immunoprecipitation after metabolic labelling of cells with [%]methionine. Two phosphorothioate 24-mers were specifically inhibitory. They targeted to the coding and noncoding region of PAI-I mRNA, respectively. The unmodified all-oxygen compounds did not influence PAIexpression in HUVECs. In summary, we present evidence that the expression elf PAI-I in HUVECs can be selectively inhihited by short antisense oligonucleotides
Recently, there has been a growing interest in the potential application of antisense oligonucleotides in the inhibition of cellular and viral gene expression. This approach is based on the assumption that short complementary DNA sequences might interact specifically with mRNA to form double-stranded helices, and so inhibit nuclear processing of the immature mRNA, transport into cytoplasm, or cytoplasmic translation into proteins. Alternatively, interaction of antisense oligonucleotides with nucleic acids may stimulate degradation of the message