Induction of the human CYP1A2 enhancer by phorbol ester

Area of Science:

• Molecular Biology
• Biochemistry
• Toxicology

Background:

• Cytochrome P4501A2 (CYP1A2) is induced by polycyclic aromatic hydrocarbons (PAHs), contributing to the bioactivation of carcinogenic amines.
• A previously identified 3-methylcholanthrene (3MC) response element is located upstream of the human CYP1A2 gene.
• This enhancer contains binding sites for the aryl hydrocarbon receptor and two sequences homologous to the phorbol 12-O-tetradecanoate 13-acetate (TPA) response element (AP-1 sites).

Purpose of the Study:

• To investigate the role of AP-1 binding sites within the CYP1A2 enhancer in gene regulation.
• To determine if tumor promoters, like TPA, can modulate CYP1A2 expression via these AP-1 sites.
• To elucidate the specific AP-1 proteins involved in the transcriptional regulation of CYP1A2.

Main Methods:

• Analysis of DNA sequences within the CYP1A2 enhancer for AP-1 consensus sequences.
• Electrophoretic mobility shift assays (EMSA) and supershift assays using nuclear extracts from TPA-treated HepG2 cells.
• Transient transfection assays in HepG2 cells using luciferase reporter constructs containing either both AP-1 sites or only the 3'AP1 site.

Main Results:

• TPA treatment increased the binding activity of AP-1 proteins (c-Jun, JunD, c-Fos) to both 5'AP1 and 3'AP1 sequences.
• Reporter gene assays showed that TPA induced luciferase activity approximately threefold with a construct containing both AP-1 sites.
• A construct with only the 3'AP1 site exhibited a sixfold induction of luciferase activity upon TPA treatment, indicating its significant role.

Conclusions:

• The human CYP1A2 gene is subject to regulation by tumor promoters through AP-1 binding sites.
• The 3'AP1 site within the CYP1A2 enhancer plays a crucial role in mediating the response to TPA.
• These findings suggest a broader regulatory mechanism for CYP1A2 induction beyond PAHs, involving tumor promoters and AP-1 signaling pathways.