Activation of serum response factor in the depolarization induction of Egr-1 transcription in pancreatic islet β-cells

Ernesto Bernal Mizrachi, B. Wice, H. Inoue, M. A. Permutt

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

The results of the current studies define the major elements whereby glucose metabolism in islet β-cells leads to transcriptional activation of an early response gene in insulinoma cell lines and in rat islets. Glucose stimulation (2-20 mM) resulted in a 4-fold increase in Egr-1 mRNA at 30 min, as did the depolarizing agents KCl and tolbutamide. This response was inhibited by diazoxide and EGTA, indicating that β-cell depolarization and Ca2+ influx, respectively, are essential. Pharmacological inhibition of the Egr-1 induction by H89 (48%) and calmidazolium (35%), but not by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 and 2 or phosphatidylinositol 3-kinase inhibitors, implied that protein kinase A and Ca2+/calmodulin pathways are involved. Deletion mapping of the Egr-1 promoter revealed that the proximal -198 base pairs containing two serum response elements (SREs) and one cAMP-response element retained the depolarization response. Depolarization resulted in phosphorylation of cAMP-response element-binding protein, yet partial inhibition by a dominant negative cAMP-response element-binding protein, along with a robust response of a cAMP-response element-mutated Egr-1 promoter suggested the presence of a second Ca2+-responsive element. Depolarization activation of 5XSRE-LUC and serum response factor (SRF)-GAL4 constructs, along with activation of SRF-GAL4 by co-transfection with constitutively active calmodulin kinase IV and protein kinase A, and binding of Ser103-phosphorylated SRF in nuclear extracts, indicated that the SRE·SRF complexes contribute to the Ca2+-mediated transcriptional regulation of Egr-1. The results of the current experiments demonstrate for the first time SRE-dependent transcription and the role of SRF, a transcription factor known to be a major component of growth responses, in glucose-mediated transcriptional regulation in insulinoma cells.

Original languageEnglish (US)
Pages (from-to)25681-25689
Number of pages9
JournalJournal of Biological Chemistry
Volume275
Issue number33
DOIs
StatePublished - Aug 18 2000
Externally publishedYes

Fingerprint

Serum Response Factor
Depolarization
Transcription
Islets of Langerhans
Serum Response Element
Chemical activation
Cyclic AMP Response Element-Binding Protein
Insulinoma
Response Elements
Cyclic AMP-Dependent Protein Kinases
Glucose
calmidazolium
Phosphatidylinositol 3-Kinase
Diazoxide
Tolbutamide
Calcium-Calmodulin-Dependent Protein Kinases
Phosphorylation
Egtazic Acid
Mitogen-Activated Protein Kinase 1
Extracellular Signal-Regulated MAP Kinases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Activation of serum response factor in the depolarization induction of Egr-1 transcription in pancreatic islet β-cells. / Bernal Mizrachi, Ernesto; Wice, B.; Inoue, H.; Permutt, M. A.

In: Journal of Biological Chemistry, Vol. 275, No. 33, 18.08.2000, p. 25681-25689.

Research output: Contribution to journalArticle

@article{2b818627bd4e4516bf8250039fd3bad9,
title = "Activation of serum response factor in the depolarization induction of Egr-1 transcription in pancreatic islet β-cells",
abstract = "The results of the current studies define the major elements whereby glucose metabolism in islet β-cells leads to transcriptional activation of an early response gene in insulinoma cell lines and in rat islets. Glucose stimulation (2-20 mM) resulted in a 4-fold increase in Egr-1 mRNA at 30 min, as did the depolarizing agents KCl and tolbutamide. This response was inhibited by diazoxide and EGTA, indicating that β-cell depolarization and Ca2+ influx, respectively, are essential. Pharmacological inhibition of the Egr-1 induction by H89 (48{\%}) and calmidazolium (35{\%}), but not by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 and 2 or phosphatidylinositol 3-kinase inhibitors, implied that protein kinase A and Ca2+/calmodulin pathways are involved. Deletion mapping of the Egr-1 promoter revealed that the proximal -198 base pairs containing two serum response elements (SREs) and one cAMP-response element retained the depolarization response. Depolarization resulted in phosphorylation of cAMP-response element-binding protein, yet partial inhibition by a dominant negative cAMP-response element-binding protein, along with a robust response of a cAMP-response element-mutated Egr-1 promoter suggested the presence of a second Ca2+-responsive element. Depolarization activation of 5XSRE-LUC and serum response factor (SRF)-GAL4 constructs, along with activation of SRF-GAL4 by co-transfection with constitutively active calmodulin kinase IV and protein kinase A, and binding of Ser103-phosphorylated SRF in nuclear extracts, indicated that the SRE·SRF complexes contribute to the Ca2+-mediated transcriptional regulation of Egr-1. The results of the current experiments demonstrate for the first time SRE-dependent transcription and the role of SRF, a transcription factor known to be a major component of growth responses, in glucose-mediated transcriptional regulation in insulinoma cells.",
author = "{Bernal Mizrachi}, Ernesto and B. Wice and H. Inoue and Permutt, {M. A.}",
year = "2000",
month = "8",
day = "18",
doi = "10.1074/jbc.M003424200",
language = "English (US)",
volume = "275",
pages = "25681--25689",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "33",

}

TY - JOUR

T1 - Activation of serum response factor in the depolarization induction of Egr-1 transcription in pancreatic islet β-cells

AU - Bernal Mizrachi, Ernesto

AU - Wice, B.

AU - Inoue, H.

AU - Permutt, M. A.

PY - 2000/8/18

Y1 - 2000/8/18

N2 - The results of the current studies define the major elements whereby glucose metabolism in islet β-cells leads to transcriptional activation of an early response gene in insulinoma cell lines and in rat islets. Glucose stimulation (2-20 mM) resulted in a 4-fold increase in Egr-1 mRNA at 30 min, as did the depolarizing agents KCl and tolbutamide. This response was inhibited by diazoxide and EGTA, indicating that β-cell depolarization and Ca2+ influx, respectively, are essential. Pharmacological inhibition of the Egr-1 induction by H89 (48%) and calmidazolium (35%), but not by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 and 2 or phosphatidylinositol 3-kinase inhibitors, implied that protein kinase A and Ca2+/calmodulin pathways are involved. Deletion mapping of the Egr-1 promoter revealed that the proximal -198 base pairs containing two serum response elements (SREs) and one cAMP-response element retained the depolarization response. Depolarization resulted in phosphorylation of cAMP-response element-binding protein, yet partial inhibition by a dominant negative cAMP-response element-binding protein, along with a robust response of a cAMP-response element-mutated Egr-1 promoter suggested the presence of a second Ca2+-responsive element. Depolarization activation of 5XSRE-LUC and serum response factor (SRF)-GAL4 constructs, along with activation of SRF-GAL4 by co-transfection with constitutively active calmodulin kinase IV and protein kinase A, and binding of Ser103-phosphorylated SRF in nuclear extracts, indicated that the SRE·SRF complexes contribute to the Ca2+-mediated transcriptional regulation of Egr-1. The results of the current experiments demonstrate for the first time SRE-dependent transcription and the role of SRF, a transcription factor known to be a major component of growth responses, in glucose-mediated transcriptional regulation in insulinoma cells.

AB - The results of the current studies define the major elements whereby glucose metabolism in islet β-cells leads to transcriptional activation of an early response gene in insulinoma cell lines and in rat islets. Glucose stimulation (2-20 mM) resulted in a 4-fold increase in Egr-1 mRNA at 30 min, as did the depolarizing agents KCl and tolbutamide. This response was inhibited by diazoxide and EGTA, indicating that β-cell depolarization and Ca2+ influx, respectively, are essential. Pharmacological inhibition of the Egr-1 induction by H89 (48%) and calmidazolium (35%), but not by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 and 2 or phosphatidylinositol 3-kinase inhibitors, implied that protein kinase A and Ca2+/calmodulin pathways are involved. Deletion mapping of the Egr-1 promoter revealed that the proximal -198 base pairs containing two serum response elements (SREs) and one cAMP-response element retained the depolarization response. Depolarization resulted in phosphorylation of cAMP-response element-binding protein, yet partial inhibition by a dominant negative cAMP-response element-binding protein, along with a robust response of a cAMP-response element-mutated Egr-1 promoter suggested the presence of a second Ca2+-responsive element. Depolarization activation of 5XSRE-LUC and serum response factor (SRF)-GAL4 constructs, along with activation of SRF-GAL4 by co-transfection with constitutively active calmodulin kinase IV and protein kinase A, and binding of Ser103-phosphorylated SRF in nuclear extracts, indicated that the SRE·SRF complexes contribute to the Ca2+-mediated transcriptional regulation of Egr-1. The results of the current experiments demonstrate for the first time SRE-dependent transcription and the role of SRF, a transcription factor known to be a major component of growth responses, in glucose-mediated transcriptional regulation in insulinoma cells.

UR - http://www.scopus.com/inward/record.url?scp=0034682785&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034682785&partnerID=8YFLogxK

U2 - 10.1074/jbc.M003424200

DO - 10.1074/jbc.M003424200

M3 - Article

VL - 275

SP - 25681

EP - 25689

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 33

ER -