Disruption of cell-cell contact maximally but transiently activates AhR-mediated transcription in 10T1/2 fibroblasts.
The aryl hydrocarbon receptor (AhR) is activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but activation without an exogenous ligand also occurs when normal cell-cell contact is prevented. Suspension of several C3H10T1/2 fibroblast clonal sub-lines that contain an integrated AhR-responsive reporter produced a time course and level of reporter activation and CYP1B1 induction that paralleled TCDD stimulation in confluent monolayer culture. Suspension activation was, however, more transient. Loss of cell-cell contact at low density also activated these reporters independent of cell cycle changes to levels comparable to TCDD stimulation of confluent cells. Loss of cell-cell contact may, therefore, activate AhR. Suspension and TCDD activations exhibited comparable nuclear translocation of AhR and then AhR/ARNT complex formation. Each AhR activation process was equally attenuated by inhibition of, respectively, HSP90 ATPase, the 26S proteosome, and by depletion of intracellular Ca2+. By contrast, the AhR antagonist alpha-naphthoflavone (alphaNF) blocked ligand-stimulated AhR activity, but not activation through loss of cell-cell contact. Suspension-induced reporter activation was selectively enhanced by LiCl, which prevented GSK-3beta effects on the simultaneously released beta-catenin. The effects of suspension and LiCl on reporters were reversed by Ro-31-8220, which did not affect beta-catenin, TCDD-activation processes, or AhR turnover. Neither LiCl nor Ro-31-8220 altered suspension-induced AhR/ARNT complex formation. Loss of cell-cell contact permits nuclear translocation and AhR activation that is largely replicated after TCDD binding, but with activity differences due to contact-sensitive factors functioning after AhR/ARNT complex formation.
Cho
Young C
YC
Molecular and Cellular Pharmacology Program, University of Wisconsin Medical School, Madison 53706, USA.
Zheng
Wenchao
W
Jefcoate
Colin R
CR
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis / Q000235:genetics; D002448:Cell Adhesion / Q000187:drug effects; D002450:Cell Communication / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000648:ultrastructure; D003470:Culture Media; D065633:Cytochrome P-450 CYP1B1; D003598:Cytoskeletal Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004785:Environmental Pollutants / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism* / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D017930:Genes, Reporter; D015151:Immunoblotting; D018021:Lithium Chloride / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D008747:Methylcellulose; D051379:Mice; D008809:Mice, Inbred C3H; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D021381:Protein Transport; D018336:Receptors, Aryl Hydrocarbon / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics*; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*; D051176:beta Catenin
C495267:CTNNB1 protein, mouse; D003470:Culture Media; D003598:Cytoskeletal Proteins; D004785:Environmental Pollutants; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D015534:Trans-Activators; D051176:beta Catenin; D008747:Methylcellulose; D008156:Luciferases; D001189:Aryl Hydrocarbon Hydroxylases; C585498:Cyp1b1 protein, mouse; D065633:Cytochrome P-450 CYP1B1; D018021:Lithium Chloride