Regulation of Cytochrome P4501A1 Gene Expression in Vascular Smooth Muscle Cells Through Aryl Hydrocarbon Receptor-Mediated Signal Transduction Requires a Protein Synthesis Inhibitor

X. L. Ou, Kenneth Ramos

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23 Citations (Scopus)

Abstract

The present studies were conducted to evaluate the pattern of cytochrome P4501A1 (CYP1A1) gene inducibility in vascular (aortic) smooth muscle cells (SMCs) upon exposure to selected aromatic hydrocarbons. Challenge of randomly cycling or synchronized subcultures of adult quail aortic SMCs with 30 μM benzo[a]pyrene (BaP) or 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h failed to induce CYP1A1 gene expression as determined by Northern analysis. However, challenge with either hydrocarbon in the presence of 10 μg/ml cycloheximide caused superinduction of CYP1A1 mRNA levels under both growth conditions. Velocity sedimentation analysis of the nuclear fraction of quail aortic SMCs treated with 10 nM [3H]-TCDD resulted in a specifically bound peak of 6.5S. Formation of the 6.5S peak was competitively inhibited by an excess of unlabeled 2,3,7,8-tetrachlorodibenzofuran (2 μM), a known aryl hydrocarbon receptor (AhR) ligand. Gel mobility shift assays of nuclear extracts from BaP- or TCDD-treated cells using a 32P-labeled Ah-responsive element consensus sequence gave ligand-inducible retarded bands. Transient transfection of the pMCAT 5.12 plasmid into SMCs followed by treatment with 30 μM BaP or 10 nM TCDD for 48 h was associated with appreciable induction of CAT activity. A comparable challenge, however, did not induce ethoxyresorufin O-deethylase activity in aortic SMCs. These results demonstrate that adult quail aortic SMCs contain the CYP1A1 gene and exhibit intact AhR-mediated signal transduction. The CYP1A1 gene is repressed under basal conditions, but treatment with cycloheximide restores constitutive expression and affords hydrocarbon inducibility. These data suggest that in adult quail aortic SMCs a labile repressor protein of CYP1A1 gene precludes transcriptional activation of the gene but does not interfere with AhR-dependent signal transduction.

Original languageEnglish (US)
Pages (from-to)116-122
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume316
Issue number1
DOIs
StatePublished - Jan 1995
Externally publishedYes

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Aryl Hydrocarbon Receptors
Signal transduction
Protein Synthesis Inhibitors
Cytochromes
Vascular Smooth Muscle
Gene expression
Smooth Muscle Myocytes
Muscle
Signal Transduction
Cells
Quail
Gene Expression
Genes
Cycloheximide
Hydrocarbons
Ligands
Repressor Proteins
Aromatic Hydrocarbons
Cytochrome P-450 CYP1A1
Benzo(a)pyrene

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

Cite this

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title = "Regulation of Cytochrome P4501A1 Gene Expression in Vascular Smooth Muscle Cells Through Aryl Hydrocarbon Receptor-Mediated Signal Transduction Requires a Protein Synthesis Inhibitor",
abstract = "The present studies were conducted to evaluate the pattern of cytochrome P4501A1 (CYP1A1) gene inducibility in vascular (aortic) smooth muscle cells (SMCs) upon exposure to selected aromatic hydrocarbons. Challenge of randomly cycling or synchronized subcultures of adult quail aortic SMCs with 30 μM benzo[a]pyrene (BaP) or 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h failed to induce CYP1A1 gene expression as determined by Northern analysis. However, challenge with either hydrocarbon in the presence of 10 μg/ml cycloheximide caused superinduction of CYP1A1 mRNA levels under both growth conditions. Velocity sedimentation analysis of the nuclear fraction of quail aortic SMCs treated with 10 nM [3H]-TCDD resulted in a specifically bound peak of 6.5S. Formation of the 6.5S peak was competitively inhibited by an excess of unlabeled 2,3,7,8-tetrachlorodibenzofuran (2 μM), a known aryl hydrocarbon receptor (AhR) ligand. Gel mobility shift assays of nuclear extracts from BaP- or TCDD-treated cells using a 32P-labeled Ah-responsive element consensus sequence gave ligand-inducible retarded bands. Transient transfection of the pMCAT 5.12 plasmid into SMCs followed by treatment with 30 μM BaP or 10 nM TCDD for 48 h was associated with appreciable induction of CAT activity. A comparable challenge, however, did not induce ethoxyresorufin O-deethylase activity in aortic SMCs. These results demonstrate that adult quail aortic SMCs contain the CYP1A1 gene and exhibit intact AhR-mediated signal transduction. The CYP1A1 gene is repressed under basal conditions, but treatment with cycloheximide restores constitutive expression and affords hydrocarbon inducibility. These data suggest that in adult quail aortic SMCs a labile repressor protein of CYP1A1 gene precludes transcriptional activation of the gene but does not interfere with AhR-dependent signal transduction.",
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N2 - The present studies were conducted to evaluate the pattern of cytochrome P4501A1 (CYP1A1) gene inducibility in vascular (aortic) smooth muscle cells (SMCs) upon exposure to selected aromatic hydrocarbons. Challenge of randomly cycling or synchronized subcultures of adult quail aortic SMCs with 30 μM benzo[a]pyrene (BaP) or 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h failed to induce CYP1A1 gene expression as determined by Northern analysis. However, challenge with either hydrocarbon in the presence of 10 μg/ml cycloheximide caused superinduction of CYP1A1 mRNA levels under both growth conditions. Velocity sedimentation analysis of the nuclear fraction of quail aortic SMCs treated with 10 nM [3H]-TCDD resulted in a specifically bound peak of 6.5S. Formation of the 6.5S peak was competitively inhibited by an excess of unlabeled 2,3,7,8-tetrachlorodibenzofuran (2 μM), a known aryl hydrocarbon receptor (AhR) ligand. Gel mobility shift assays of nuclear extracts from BaP- or TCDD-treated cells using a 32P-labeled Ah-responsive element consensus sequence gave ligand-inducible retarded bands. Transient transfection of the pMCAT 5.12 plasmid into SMCs followed by treatment with 30 μM BaP or 10 nM TCDD for 48 h was associated with appreciable induction of CAT activity. A comparable challenge, however, did not induce ethoxyresorufin O-deethylase activity in aortic SMCs. These results demonstrate that adult quail aortic SMCs contain the CYP1A1 gene and exhibit intact AhR-mediated signal transduction. The CYP1A1 gene is repressed under basal conditions, but treatment with cycloheximide restores constitutive expression and affords hydrocarbon inducibility. These data suggest that in adult quail aortic SMCs a labile repressor protein of CYP1A1 gene precludes transcriptional activation of the gene but does not interfere with AhR-dependent signal transduction.

AB - The present studies were conducted to evaluate the pattern of cytochrome P4501A1 (CYP1A1) gene inducibility in vascular (aortic) smooth muscle cells (SMCs) upon exposure to selected aromatic hydrocarbons. Challenge of randomly cycling or synchronized subcultures of adult quail aortic SMCs with 30 μM benzo[a]pyrene (BaP) or 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h failed to induce CYP1A1 gene expression as determined by Northern analysis. However, challenge with either hydrocarbon in the presence of 10 μg/ml cycloheximide caused superinduction of CYP1A1 mRNA levels under both growth conditions. Velocity sedimentation analysis of the nuclear fraction of quail aortic SMCs treated with 10 nM [3H]-TCDD resulted in a specifically bound peak of 6.5S. Formation of the 6.5S peak was competitively inhibited by an excess of unlabeled 2,3,7,8-tetrachlorodibenzofuran (2 μM), a known aryl hydrocarbon receptor (AhR) ligand. Gel mobility shift assays of nuclear extracts from BaP- or TCDD-treated cells using a 32P-labeled Ah-responsive element consensus sequence gave ligand-inducible retarded bands. Transient transfection of the pMCAT 5.12 plasmid into SMCs followed by treatment with 30 μM BaP or 10 nM TCDD for 48 h was associated with appreciable induction of CAT activity. A comparable challenge, however, did not induce ethoxyresorufin O-deethylase activity in aortic SMCs. These results demonstrate that adult quail aortic SMCs contain the CYP1A1 gene and exhibit intact AhR-mediated signal transduction. The CYP1A1 gene is repressed under basal conditions, but treatment with cycloheximide restores constitutive expression and affords hydrocarbon inducibility. These data suggest that in adult quail aortic SMCs a labile repressor protein of CYP1A1 gene precludes transcriptional activation of the gene but does not interfere with AhR-dependent signal transduction.

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