Dexamethasone represses phorbol ester-, forskolin-, and calcium-stimulated expression of a preproenkephalin A promoter-chloramphenicol acetyltransferase gene via a receptor-mediated mechanism

David Crabb, Mark J. Stewart, Rebecca Chan

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

CV-1 cells were stably transfected with a preproenkephalin A (PPE) promoter-chloramphenicol acetyltransferase (CAT)reporter plasmid containing -176 to +171 bp of the human PPE gene. Low levels of CAT were expressed constitutively. The reporter enzyme activity was induced by treatment of the cells for 6 h with drugs that increased intracellular cAMP (forskolin and 8-bromo-cAMP), intracellular calcium (A23187), or protein kinase C activity (tetradecanoyl phorbol-4-acetate, TPA) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Co-administration of dexamethasone reduced the magnitude of phorbol ester-stimulated CAT activity by about 50%, while there were smaller but not significant effects on forskolin- or A23187-stimulated expression of this reporter construct. In transient transfections which included the PPE-CAT reporter gene and a glucocorticoid receptor expression plasmid, dexamethasone significantly reduced stimulated expression of the reporter by TPA, forskolin, and A23187. The effect was observed with 10-8-10-6 M dexamethasone and was blocked by the presence of the glucocorticoid antagonist Ru486, suggesting that the effect of dexamethasone was mediated by the glucocorticoid receptor. The promoter region contained in this construct lacks a classical glucocorticoid response element or known negative elements; thus, dexamethasone may reduce stimulated expression of the PPE promoter via indirect effects.

Original languageEnglish
Pages (from-to)103-108
Number of pages6
JournalMolecular Brain Research
Volume42
Issue number1
DOIs
StatePublished - Nov 1996

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Chloramphenicol O-Acetyltransferase
Phorbol Esters
Colforsin
Dexamethasone
Calcium
Calcimycin
Genes
Glucocorticoid Receptors
Glucocorticoids
Acetates
Plasmids
8-Bromo Cyclic Adenosine Monophosphate
Phosphodiesterase Inhibitors
Response Elements
Reporter Genes
Genetic Promoter Regions
Protein Kinase C
Transfection
preproenkephalin
Enzymes

Keywords

  • Enkephalin
  • Glucocorticoid
  • Hormone
  • Transcription
  • Transfection

ASJC Scopus subject areas

  • Molecular Biology
  • Cellular and Molecular Neuroscience

Cite this

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title = "Dexamethasone represses phorbol ester-, forskolin-, and calcium-stimulated expression of a preproenkephalin A promoter-chloramphenicol acetyltransferase gene via a receptor-mediated mechanism",
abstract = "CV-1 cells were stably transfected with a preproenkephalin A (PPE) promoter-chloramphenicol acetyltransferase (CAT)reporter plasmid containing -176 to +171 bp of the human PPE gene. Low levels of CAT were expressed constitutively. The reporter enzyme activity was induced by treatment of the cells for 6 h with drugs that increased intracellular cAMP (forskolin and 8-bromo-cAMP), intracellular calcium (A23187), or protein kinase C activity (tetradecanoyl phorbol-4-acetate, TPA) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Co-administration of dexamethasone reduced the magnitude of phorbol ester-stimulated CAT activity by about 50{\%}, while there were smaller but not significant effects on forskolin- or A23187-stimulated expression of this reporter construct. In transient transfections which included the PPE-CAT reporter gene and a glucocorticoid receptor expression plasmid, dexamethasone significantly reduced stimulated expression of the reporter by TPA, forskolin, and A23187. The effect was observed with 10-8-10-6 M dexamethasone and was blocked by the presence of the glucocorticoid antagonist Ru486, suggesting that the effect of dexamethasone was mediated by the glucocorticoid receptor. The promoter region contained in this construct lacks a classical glucocorticoid response element or known negative elements; thus, dexamethasone may reduce stimulated expression of the PPE promoter via indirect effects.",
keywords = "Enkephalin, Glucocorticoid, Hormone, Transcription, Transfection",
author = "David Crabb and Stewart, {Mark J.} and Rebecca Chan",
year = "1996",
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T1 - Dexamethasone represses phorbol ester-, forskolin-, and calcium-stimulated expression of a preproenkephalin A promoter-chloramphenicol acetyltransferase gene via a receptor-mediated mechanism

AU - Crabb, David

AU - Stewart, Mark J.

AU - Chan, Rebecca

PY - 1996/11

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N2 - CV-1 cells were stably transfected with a preproenkephalin A (PPE) promoter-chloramphenicol acetyltransferase (CAT)reporter plasmid containing -176 to +171 bp of the human PPE gene. Low levels of CAT were expressed constitutively. The reporter enzyme activity was induced by treatment of the cells for 6 h with drugs that increased intracellular cAMP (forskolin and 8-bromo-cAMP), intracellular calcium (A23187), or protein kinase C activity (tetradecanoyl phorbol-4-acetate, TPA) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Co-administration of dexamethasone reduced the magnitude of phorbol ester-stimulated CAT activity by about 50%, while there were smaller but not significant effects on forskolin- or A23187-stimulated expression of this reporter construct. In transient transfections which included the PPE-CAT reporter gene and a glucocorticoid receptor expression plasmid, dexamethasone significantly reduced stimulated expression of the reporter by TPA, forskolin, and A23187. The effect was observed with 10-8-10-6 M dexamethasone and was blocked by the presence of the glucocorticoid antagonist Ru486, suggesting that the effect of dexamethasone was mediated by the glucocorticoid receptor. The promoter region contained in this construct lacks a classical glucocorticoid response element or known negative elements; thus, dexamethasone may reduce stimulated expression of the PPE promoter via indirect effects.

AB - CV-1 cells were stably transfected with a preproenkephalin A (PPE) promoter-chloramphenicol acetyltransferase (CAT)reporter plasmid containing -176 to +171 bp of the human PPE gene. Low levels of CAT were expressed constitutively. The reporter enzyme activity was induced by treatment of the cells for 6 h with drugs that increased intracellular cAMP (forskolin and 8-bromo-cAMP), intracellular calcium (A23187), or protein kinase C activity (tetradecanoyl phorbol-4-acetate, TPA) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Co-administration of dexamethasone reduced the magnitude of phorbol ester-stimulated CAT activity by about 50%, while there were smaller but not significant effects on forskolin- or A23187-stimulated expression of this reporter construct. In transient transfections which included the PPE-CAT reporter gene and a glucocorticoid receptor expression plasmid, dexamethasone significantly reduced stimulated expression of the reporter by TPA, forskolin, and A23187. The effect was observed with 10-8-10-6 M dexamethasone and was blocked by the presence of the glucocorticoid antagonist Ru486, suggesting that the effect of dexamethasone was mediated by the glucocorticoid receptor. The promoter region contained in this construct lacks a classical glucocorticoid response element or known negative elements; thus, dexamethasone may reduce stimulated expression of the PPE promoter via indirect effects.

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