INVESTIGADORES
CERUTI Julieta Maria
artículos
Título:
Hepatic Nuclear Factor 3 and Nuclear Factor 1 Regulate
Autor/es:
MARÍA E SCASSA; ALEJANDRA S GUBERMAN; JULIETA M CERUTI; EDUARDO T CÁNEPA
Revista:
JOURNAL OF BIOLOGICAL CHEMISTRY
Editorial:
The American Society for Biochemistry and Molecular Biology, Inc.
Referencias:
Año: 2004 vol. 279 p. 28082 - 28092
ISSN:
0021-9258
Resumen:
Although the negative regulation of gene expression
by insulin has been widely studied, the transcription
factors responsible for the insulin effect are still unknown.
The purpose of this work was to explore the
molecular mechanisms involved in the insulin repression
of the 5-aminolevulinate synthase (ALAS) gene. Deletion
analysis of the 5-regulatory region allowed us to
identify an insulin-responsive region located at 459 to-regulatory region allowed us to
identify an insulin-responsive region located at 459 to459 to
354 bp. This fragment contains a highly homologous
insulin-responsive (IRE) sequence. By transient transfection
assays, we determined that hepatic nuclear factor
3 (HNF3) and nuclear factor 1 (NF1) are necessary
for an appropriate expression of the ALAS gene. Insulin
overrides the HNF3 or HNF3 plus NF1-mediated stimulation
of ALAS transcriptional activity. Electrophoretic
mobility shift assay and Southwestern blotting
indicate that HNF3 binds to the ALAS promoter. Mutational
analysis of this region revealed that IRE disruption
abrogates insulin action, whereas mutation of the
HNF3 element maintains hormone responsiveness. This
dissociation between HNF3 binding and insulin action
suggests that HNF3 is not the sole physiologic mediator
of insulin-induced transcriptional repression. Furthermore,
Southwestern blotting assay shows that at
least two polypeptides other than HNF3 can bind to
ALAS promoter and that this binding is dependent on
the integrity of the IRE. We propose a model in which
insulin exerts its negative effect through the disturbance
of HNF3 binding or transactivation potential,
probably due to specific phosphorylation of this transcription
factor by Akt. In this regard, results obtained
from transfection experiments using kinase inhibitors
support this hypothesis. Due to this event, NF1 would
lose accessibility to the promoter. The posttranslational
modification of HNF3 would allow the binding of a protein
complex that recognizes the core IRE. These results
provide a potential mechanism for the insulin-mediated
repression of IRE-containing promoters.354 bp. This fragment contains a highly homologous
insulin-responsive (IRE) sequence. By transient transfection
assays, we determined that hepatic nuclear factor
3 (HNF3) and nuclear factor 1 (NF1) are necessary
for an appropriate expression of the ALAS gene. Insulin
overrides the HNF3 or HNF3 plus NF1-mediated stimulation
of ALAS transcriptional activity. Electrophoretic
mobility shift assay and Southwestern blotting
indicate that HNF3 binds to the ALAS promoter. Mutational
analysis of this region revealed that IRE disruption
abrogates insulin action, whereas mutation of the
HNF3 element maintains hormone responsiveness. This
dissociation between HNF3 binding and insulin action
suggests that HNF3 is not the sole physiologic mediator
of insulin-induced transcriptional repression. Furthermore,
Southwestern blotting assay shows that at
least two polypeptides other than HNF3 can bind to
ALAS promoter and that this binding is dependent on
the integrity of the IRE. We propose a model in which
insulin exerts its negative effect through the disturbance
of HNF3 binding or transactivation potential,
probably due to specific phosphorylation of this transcription
factor by Akt. In this regard, results obtained
from transfection experiments using kinase inhibitors
support this hypothesis. Due to this event, NF1 would
lose accessibility to the promoter. The posttranslational
modification of HNF3 would allow the binding of a protein
complex that recognizes the core IRE. These results
provide a potential mechanism for the insulin-mediated
repression of IRE-containing promoters. or HNF3 plus NF1-mediated stimulation
of ALAS transcriptional activity. Electrophoretic
mobility shift assay and Southwestern blotting
indicate that HNF3 binds to the ALAS promoter. Mutational
analysis of this region revealed that IRE disruption
abrogates insulin action, whereas mutation of the
HNF3 element maintains hormone responsiveness. This
dissociation between HNF3 binding and insulin action
suggests that HNF3 is not the sole physiologic mediator
of insulin-induced transcriptional repression. Furthermore,
Southwestern blotting assay shows that at
least two polypeptides other than HNF3 can bind to
ALAS promoter and that this binding is dependent on
the integrity of the IRE. We propose a model in which
insulin exerts its negative effect through the disturbance
of HNF3 binding or transactivation potential,
probably due to specific phosphorylation of this transcription
factor by Akt. In this regard, results obtained
from transfection experiments using kinase inhibitors
support this hypothesis. Due to this event, NF1 would
lose accessibility to the promoter. The posttranslational
modification of HNF3 would allow the binding of a protein
complex that recognizes the core IRE. These results
provide a potential mechanism for the insulin-mediated
repression of IRE-containing promoters. is not the sole physiologic mediator
of insulin-induced transcriptional repression. Furthermore,
Southwestern blotting assay shows that at
least two polypeptides other than HNF3 can bind to
ALAS promoter and that this binding is dependent on
the integrity of the IRE. We propose a model in which
insulin exerts its negative effect through the disturbance
of HNF3 binding or transactivation potential,
probably due to specific phosphorylation of this transcription
factor by Akt. In this regard, results obtained
from transfection experiments using kinase inhibitors
support this hypothesis. Due to this event, NF1 would
lose accessibility to the promoter. The posttranslational
modification of HNF3 would allow the binding of a protein
complex that recognizes the core IRE. These results
provide a potential mechanism for the insulin-mediated
repression of IRE-containing promoters. can bind to
ALAS promoter and that this binding is dependent on
the integrity of the IRE. We propose a model in which
insulin exerts its negative effect through the disturbance
of HNF3 binding or transactivation potential,
probably due to specific phosphorylation of this transcription
factor by Akt. In this regard, results obtained
from transfection experiments using kinase inhibitors
support this hypothesis. Due to this event, NF1 would
lose accessibility to the promoter. The posttranslational
modification of HNF3 would allow the binding of a protein
complex that recognizes the core IRE. These results
provide a potential mechanism for the insulin-mediated
repression of IRE-containing promoters. binding or transactivation potential,
probably due to specific phosphorylation of this transcription
factor by Akt. In this regard, results obtained
from transfection experiments using kinase inhibitors
support this hypothesis. Due to this event, NF1 would
lose accessibility to the promoter. The posttranslational
modification of HNF3 would allow the binding of a protein
complex that recognizes the core IRE. These results
provide a potential mechanism for the insulin-mediated
repression of IRE-containing promoters.