Suspension-mediated induction of Hepa 1c1c7 Cyp1a-1 expression is dependent on the Ah receptor signal transduction pathway.
We have recently demonstrated that release of normal human epithelial cells from cell-substratum and/or cell-cell adhesion generates cellular signals that induce the expression of CYP1A1 in the absence of xenobiotic polycyclic aromatic hydrocarbons (Sadek, C. M., and Allen-Hoffmann, B. L. (1994) J. Biol. Chem. 169, 16067-16074). To directly test the involvement of the Ah receptor signal transduction pathway in CYP1A1 induction following suspension of epithelial cells, we analyzed wild-type Hepa 1c1c7 cells, a subclone of the Hepa-1c1 mouse hepatoma line, and two mutant Hepa 1c1c7 lines, Class I and Class II. Suspension of wild-type Hepa 1c1c7 cells for 4 h led to an induction of steady state levels of CYP1A1 mRNA, similar to that obtained following treatment of adherent cells with 10(-9) M 2,3,7,8-tetrachlorodibenzo-p-dioxin. Mutants of the Hepa 1c1c7 cells defective in different aspects of the Ah receptor signal transduction pathway exhibited negligible (Class I) or no (Class II) suspension-mediated induction of CYP1A1 mRNA. Gel mobility shift analysis of nuclear extracts from suspended or 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated wild-type cells showed that both treatments produced identical shifts in the mobility of an XRE-containing probe. Antibody supershift experiments confirmed that the Ah receptor was a component of the DNA-protein complex from suspended wild-type Hepa 1c1c7 cells. These data directly demonstrate that suspension of wild-type Hepa 1c1c7 cells leads to nuclear localization and activation of the Ah receptor to a DNA-binding form.
Sadek
C M
CM
Department of Pathology, University of Wisconsin, Madison 53706.
Allen-Hoffmann
B L
BL
D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D002448:Cell Adhesion; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004790:Enzyme Induction; D005786:Gene Expression Regulation / Q000187:drug effects; D066298:In Vitro Techniques; D008114:Liver Neoplasms, Experimental / Q000201:enzymology / Q000473:pathology*; D051379:Mice; D010088:Oxidoreductases / Q000378:metabolism*; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D018336:Receptors, Aryl Hydrocarbon / Q000502:physiology*; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000201:enzymology
C497397:Arnt protein, mouse; D004268:DNA-Binding Proteins; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1