Abstract
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor with constitutive activities and those induced by xenobiotic ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). One unexplained cellular role for the AHR is its ability to promote cell cycle progression in the absence of exogenous ligands, whereas treatment with exogenous ligands induces cell cycle arrest. Within the cell cycle, progression from G1 to S phase is controlled by sequential phosphorylation of the retinoblastoma protein (RB1) by cyclin D–cyclin-dependent kinase (CDK) 4/6 complexes. In this study, the functional interactions between the AHR, CDK4, and cyclin D1 (CCND1) were investigated as a potential mechanism for the cell cycle regulation by the AHR. Time course cell cycle and molecular experiments were performed in human breast cancer cells. The results demonstrated that the AHR and CDK4 interact within the cell cycle, and the interaction was disrupted upon TCDD treatment. The disruption was temporally correlated with G1 cell cycle arrest and decreased phosphorylation of RB1. Biochemical reconstitution assays using in vitro-translated protein recapitulated the AHR and CDK4 interaction and showed that CCND1 was also part of the complex. In vitro assays for CDK4 kinase activity demonstrated that RB1 phosphorylation by the AHR/CDK4/CCND1 complex was reduced in the presence of TCDD. The results suggest that the AHR interacts in a complex with CDK4 and CCND1 in the absence of exogenous ligands to facilitate cell cycle progression. This interaction is disrupted by exogenous ligands, such as TCDD, to induce G1 cell cycle arrest.
Footnotes
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This work was supported by The Hamner Pilot Projects Initiative, The Hamner Institutes for Health Sciences.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.059675.
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ABBREVIATIONS:
- AHR
- aryl hydrocarbon receptor
- ARNT
- aryl hydrocarbon nuclear translocator
- CDK4
- cyclin-dependent kinase 4
- ER−
- estrogen receptor negative
- ER+
- estrogen receptor positive
- TCDD
- 2,3,7,8-tetrachlorodibenzo-p-dioxin
- RB1
- retinoblastoma protein
- CCND1
- cyclin D1
- FBS
- fetal bovine serum
- C/D FBS
- charcoal-stripped fetal bovine serum
- E2
- 17-β-estradiol
- HSP90
- 90-kDa heat shock protein
- DMSO
- dimethyl sulfoxide
- DMEM
- Dulbecco's modified Eagle's medium
- PAGE
- polyacrylamide gel electrophoresis
- PVDF
- polyvinylidene difluoride
- PCR
- polymerase chain reaction
- IP
- immunoprecipitation
- ECL
- enhanced chemiluminescence
- MEM
- minimal essential medium.
- Received July 22, 2009.
- Accepted November 16, 2009.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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