UVM Theses and Dissertations
Format:
Print
Author:
Burch, Peter Matthew
Dept./Program:
Cell and Molecular Biology Program
Year:
2004
Degree:
Ph. D.
Abstract:
Mitogens activate cell signaling and gene expression cascades that culminate in expression of cyclin D1 during the G0 to G1 transition of the cell cycle. Using cell cycle arrest in response to oxidative stress, we have delineated a dynamic program of chromatin trafficking of c-Fos and Fra-1 required for cyclin D1 expression during cell cycle re-entry. In serum-stimulated lung epithelial cells, c-Fos was expressed, recruited to chromatin, phosphorylated at ERK1,2-dependent sites and degraded prior to prolonged recruitment of Fra-1 to chromatin. Immunostaining showed that expression of nuclear c-Fos and cyclin D1 are mutually exclusive, whereas nuclear Fra-1 and cyclin D1 are coexpressed as cells traverse G1. Oxidative stress prolonged the accumulation of phospho-ERK1,2 and phospho-c-Fos on chromatin, inhibited entry of Fra-1 into the nucleus, and blocked cyclin D1 expression. After induction of the immediate early gene response in the presence of oxidative stress, inhibition of ERK1,2 signaling promoted degradation of c-Fos, recruitment of Fra-1 to chromatin and expression of cyclin D1. Our data indicate termination of nuclear ERK1,2 signaling is required for an exchange of Fra-1 for c-Fos on chromatin and initiation of cyclin D1 expression at the G0 to G1 transition of the cell cycle.
To better understand the mechanism by which oxidant exposure during cell cycle re-entry could result in the accumulation of phospho-ERK 1,2 and phospho-c-Fos on chromatin, the role of nuclear export was investigated. Cell cycle re-entry is accompanied by a dynamic program of protein trafficking that results in the nuclear localization of signaling proteins and transcription factors. Using RanBP1 as a marker of nuclear export, we show here that cells exposed to oxidative stress during cell cycle re-entry exhibit a dose-dependent inhibition of Crm1-dependent nuclear export. Collapse of the RanBP1 gradient by oxidative stress was associated with accumulation of Crm1, phospho-ERK, and phospho-c-Fos on chromatin. Treatment with leptomycin B, a specific inhibitor of Crm1-dependent nuclear export, prolonged accumulation of phosphorylated c-Fos on chromatin during cell cycle reentry without activating ERK, suggesting Crm1 is required for both dissociation from chromatin and export of phospho-c-Fos during the G0 to G1 transition of the cell cycle. Our results indicate Crm1-mediated nuclear export is one facet a dynamic program of subcellular trafficking of transcription factors required for cell cycle progression in response to mitogens.
To better understand the mechanism by which oxidant exposure during cell cycle re-entry could result in the accumulation of phospho-ERK 1,2 and phospho-c-Fos on chromatin, the role of nuclear export was investigated. Cell cycle re-entry is accompanied by a dynamic program of protein trafficking that results in the nuclear localization of signaling proteins and transcription factors. Using RanBP1 as a marker of nuclear export, we show here that cells exposed to oxidative stress during cell cycle re-entry exhibit a dose-dependent inhibition of Crm1-dependent nuclear export. Collapse of the RanBP1 gradient by oxidative stress was associated with accumulation of Crm1, phospho-ERK, and phospho-c-Fos on chromatin. Treatment with leptomycin B, a specific inhibitor of Crm1-dependent nuclear export, prolonged accumulation of phosphorylated c-Fos on chromatin during cell cycle reentry without activating ERK, suggesting Crm1 is required for both dissociation from chromatin and export of phospho-c-Fos during the G0 to G1 transition of the cell cycle. Our results indicate Crm1-mediated nuclear export is one facet a dynamic program of subcellular trafficking of transcription factors required for cell cycle progression in response to mitogens.