Proteasome inhibition induces nuclear translocation and transcriptional activation of the dioxin receptor in mouse embryo primary fibroblasts in the absence of xenobiotics.
The aryl hydrocarbon receptor (AHR) is a transcription factor that is highly conserved during evolution and shares important structural features with the Drosophila developmental regulators Sim and Per. Although much is known about the mechanism of AHR activation by xenobiotics, little information is available regarding its activation by endogenous stimuli in the absence of exogenous ligand. In this study, using embryonic primary fibroblasts, we have analyzed the role of proteasome inhibition on AHR transcriptional activation in the absence of xenobiotics. Proteasome inhibition markedly reduced cytosolic AHR without affecting its total cellular content. Cytosolic AHR depletion was the result of receptor translocation into the nuclear compartment, as shown by transient transfection of a green fluorescent protein-tagged AHR and by immunoblot analysis of nuclear extracts. Gel retardation experiments showed that proteasome inhibition induced transcriptionally active AHR-ARNT heterodimers able to bind to a consensus xenobiotic-responsive element. Furthermore, nuclear AHR was transcriptionally active in vivo, as shown by the induction of the endogenous target gene CYP1A2. Synchronized to AHR activation, proteasome inhibition also induced a transient increase in AHR nuclear translocator (ARNT) at the protein and mRNA levels. Since nuclear levels of AHR and ARNT are relevant for AHR transcriptional activation, our data suggest that proteasome inhibition, through a transient increase in ARNT expression, could promote AHR stabilization and accumulation into the nuclear compartment. An elevated content of nuclear AHR could favor AHR-ARNT heterodimers able to bind to xenobiotic-responsive elements and to induce gene transcription in the absence of xenobiotics. Thus, depending on the cellular context, physiologically regulated proteasome activity could participate in the control of endogenous AHR functions.
Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain.
B
B
Santiago-Josefat
E
E
Pozo-Guisado
S
S
Mulero-Navarro
P M
PM
Fernandez-Salguero
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus; D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D046988:Proteasome Endopeptidase Complex; D012333:RNA, Messenger / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D008024:Ligands; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019388:Cytochrome P-450 CYP1A2; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex